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activation of the sympathetic nervous system exerts a number of physiological responses either directly through stimulation of postganglionic sympathetic nerves localised in target organs or indirectly through the activation of powerful humoral systems. while the importance of sympathetic tone is readily acknowledged for cardiovascular and blood pressure regulation it is less well appreciated that activation of the sympathetic nervous system forms an integral part of energy homeostasis and can exert profound metabolic effects. insufficient physical activity and excess energy intake, coupled with genetic programming, have been attributed to the rising incidence of obesity, hypertension, dyslipidemia, insulin resistance, and hyperglycemia in western societies. more importantly, the clustering of these metabolic conditions, referred to as the metabolic syndrome (mets), has become increasingly more common with one - quarter of all adults predicted to have mets. given the augmented risk for type 2 diabetes, cardiovascular disease, and premature mortality associated with the mets [24 ] there is growing interest in understanding the complex pathways underlying the metabolic derangements seen in the condition. accumulating data from animal and human studies suggest that central sympathetic activity plays a pivotal role in the aetiology and complications of mets and its associated conditions. this is evidenced by a distinct preponderance for patients with the mets to exhibit clinical signs of chronic sympathoexcitation such as elevated urinary noradrenaline levels, increased efferent muscle sympathetic nerve activity, and elevated rates of plasma noradrenaline spillover, even in the absence of hypertension [59 ]. interestingly, recent evidence indicates that the benefits derived from diet and exercise induced weight loss are in part mediated by a reduction in sympathetic nerve activity. latest findings from clinical studies suggest pharmacological and device - based therapies (i.e., imidazoline agonists and catheter - based renal denervation) that specifically target central sympathetic outflow may also assist in improving metabolic control in these subjects. the purpose of this review is to present an overview of the evidence supporting the role of the sympathetic nervous system in metabolic control. this review will also focus on the possible mechanisms linking central sympathetic overactivity to the pathophysiology of the mets, with particular emphasis on the putative role of the sympathetic nervous system in two key metabolic alterations, central obesity and insulin resistance. it will conclude by highlighting some of the emerging therapeutic options available for the treatment of mets - related conditions that specifically target a reduction in central sympathetic activity. central sympathetic outflow is principally driven by a network of neurons located in the rostral ventrolateral medulla. these neurons provide excitatory output to preganglionic neurons located in the intermediolateral cell column of the spinal cord that innervate several target organs through postganglionic sympathetic fibers. excitatory drive can be intrinsically generated, chemically mediated, or differentially controlled via the cortical, limbic, and midbrain regions of the central nervous system. a number of afferent inputs from peripheral reflexes (i.e., arterial baroreflexes, chemoreceptors, and hormonal mediators) can stimulate the rostral ventrolateral medulla and alter sympathetic nerve activity via neurons that terminate in the nucleus tractus solitarius of the medulla oblongata. additionally, circulating factors that are able to cross the blood - brain barrier via circumventricular organs can also influence central sympathetic outflow. while the neuroanatomical interactions that govern the sympathetic nervous system are yet to be fully elucidated, sympathetic tone is recognised as an important mediator of cardiovascular function predominantly through its direct effects on beta - adrenergic receptors in the heart to modulate cardiac output and on alpha - adrenergic receptors in blood vessels to modulate arteriolar resistance and venous capacitance. activation of adrenergic receptors in the kidney is also important for modulating blood pressure as neuronal noradrenaline promotes the release of renin from the juxtaglomerular apparatus, sodium retention, and vasoconstriction. in terms of metabolism, the sympathetic nervous system is fundamental in controlling daily energy expenditure via the regulation of resting metabolic rate and initiation of thermogenesis in response to physiologically relevant stimuli, that is, changing energy states, food intake, carbohydrate consumption, hyperinsulinemia, and exposure to cold. activation of sympathetic nerves innervating the liver, pancreas, skeletal muscle, and adipose tissue can also elicit acute catabolic responses (i.e., glycogenolysis and lipolysis). it is important to note that not all organs are targeted equally by the sympathetic nervous system with the metabolic effects ensuing from increased central sympathetic outflow dependent on the adrenergic receptors present in the target organ, the number of neurons recruited, and whether an individual is in a fasted or postprandial state. as shown in figure 1, acute activation of splanchnic sympathetic nerves innervating parenchymal cells in the liver is shown to produce a rapid and marked production of glucose following a meal but promotes gluconeogenesis when fasted. activation of the adrenal medulla can also stimulate the release of catecholamines to further promote hepatic glucose production. in the pancreas, activation of splanchnic sympathetic nerves promotes glucagon secretion via beta - adrenergic receptors on islet cells, which is secondary to the inhibition of insulin secretion via alpha - adrenergic receptor activation. sympathetic nerves innervating skeletal muscle can modulate glucose uptake independent of a concomitant increase in plasma insulin levels via activation of beta - adrenergic receptors using camp as the second messenger. conversely, neuronal stimulation of alpha - adrenergic receptors in arterioles, which elicits vasoconstriction, impairs glucose uptake in skeletal muscle. compared to the liver, pancreas, and skeletal muscle, which are also under parasympathetic control, adipose tissue central sympathetic outflow directly stimulates adipocyte lipolysis by binding to beta - adrenergic receptors in white adipose tissue to activate camp - dependent pathways to translocate inactive lipase. noradrenaline has also been shown to decrease the uptake of triglyceride - rich lipoprotein into white adipose tissue via the inhibition of the rate limiting enzyme lipoprotein lipase (lpl), while beta - adrenergic activation in skeletal muscle stimulates lpl activity and promotes lipid uptake. due to its ability to express various adipokines, white adipose tissue can also regulate sympathetic output through the production of centrally acting peptide hormones. of those adipokines that are secreted from adipose tissue and modulate central sympathetic activity, leptin is the best described. it is able to cross the blood - brain barrier to act directly on leptin receptors in higher brain regions involved in the regulation of sympathetic tone. in brown adipose tissue, direct neuronal activation of beta - adrenergic receptors during exposure to different environmental stimuli (i.e., exposure to cold) while the metabolic processes that ensue from acute sympathetic activation may be desirable under certain circumstances, it is clear that chronic stimulation of the sympathetic nervous system has the potential to augment risk for mets, through the development of obesity, hyperglycaemia, insulin resistance, and hypertension. the mets is a constellation of metabolic abnormalities characterized by central (abdominal) obesity, insulin resistance, hyperglycemia, dyslipidemia, hypertension, and systemic inflammation that confers significant risk for the development of both type 2 diabetes and cardiovascular disease. central obesity is considered the cardinal feature of mets with several major organizations recognizing waist circumference as the primary screening tool for mets. it is notable that subjects with central obesity often have more metabolic disorders and consequently are at greater risk of developing diabetes than those without central obesity. prediabetes is defined by impaired fasting glucose and/or impaired glucose tolerance with a glycosylated hemoglobin level (hba1c) between 5.7 and 6.4% [14, 15 ]. given that hyperglycemia is a component of the mets and most individuals with mets are insulin - resistant, it is often considered a prediabetic state. the observation that older, nondiabetic adults are two times more likely to have the mets with hyperglycemia than hyperglycemia only confirms an overlap between the conditions. the mets is associated with a 5-fold increased risk for diabetes. while most assume this to be driven by hyperglycemia, mets without prediabetes carries a similar level of risk. one of the reasons why the mets is as strong a predictor of diabetes with or without prediabetes is the presence of central obesity which not only acts to promote insulin resistance but can potentiate beta cell dysfunction through lipotoxicity. there is substantial evidence in support of the sympathetic nervous system being exceedingly active in individuals with the mets and its key metabolic alterations, central obesity, and insulin resistance. compared to healthy lean individuals, obese adults are consistently shown to have raised urinary noradrenaline and metabolite levels and elevated rates of whole - body noradrenaline spillover in plasma. furthermore, direct sympathetic nerve recording using microneurography demonstrates that obese individuals display increased resting sympathetic outflow to skeletal muscle [6, 22, 23 ]. interestingly, the degree of sympathetic overactivity observed in obese individuals appears to be dependent on body fat distribution with central obesity shown to be associated with greater sympathetic nerve activation than subcutaneous obesity [7, 24 ]. the link between obesity and sympathetic overactivity is further strengthened by the observation that weight loss in obese individuals causes a marked decrease in muscle sympathetic nerve activity [6, 25 ] and increase in muscle sympathetic nerve activity following weight gain. after a meal, the normal physiological response in healthy humans is to increase sympathetic nerve activity, as indicated by a rise in plasma noradrenaline concentrations and increase in muscle sympathetic nerve activity. increased central sympathetic activity in the postprandial state is important to not only promote thermogenesis but induce compensatory peripheral vasoconstriction to maintain blood pressure following splanchnic vasodilatation. insulin - resistant individuals, particularly those whose insulin resistance is associated with central adiposity, display blunted sympathetic neuronal responses to physiological hyperinsulinemia, glucose consumption, and changes in energy states. indeed, straznicky. confirmed that insulin - resistant subjects display a blunted sympathetic neural response to glucose ingestion compared with age- and blood pressure - matched insulin - sensitive subjects, despite a 2-fold greater increase in plasma insulin concentrations. evidence for sympathetic overactivity being linked to the onset of diabetes comes from several long - term prospective cohort studies that show both baseline noradrenaline levels and sympathetic reactivity predict future risk of insulin resistance and diabetes [2931 ]. experimental studies in patients with type 2 diabetes show significantly augmented resting muscle sympathetic nerve activity compared to individuals with prediabetes or obesity. blunted sympathetic responsiveness to glucose and elevated arterial noradrenaline levels are also more exaggerated in treatment naive type 2 diabetics compared to individuals with impaired glucose tolerance suggesting that the progression from prediabetes to diabetes is characterized by profound disturbances in central sympathetic nerve activity. several pathophysiological mechanisms linking central sympathetic overactivity with the mets and its core components of central obesity and insulin resistance have been proposed with ongoing debate as to whether sympathetic overactivity is a consequence or a cause of metabolic dysfunction. landsberg first proposed a link between the development of metabolic abnormalities and the sympathetic nervous system 25 years ago when he hypothesised that elevated circulating insulin levels, resulting from insulin resistance associated with obesity, caused an elevation in central sympathetic activity, which precipitated the development of hypertension. his hypothesis was based on observations in rodents that showed that overfeeding leads to an increase in sympathetic nerve activity and rise in blood pressure. subsequent studies in healthy adults confirmed this occurrence with the infusion of insulin, in the presence of stable glucose levels, shown to increase muscle sympathetic nerve activity independent of insulin 's vasodilatory effects. these include direct activation of the sympathetic nervous system by higher cerebral nuclei during overfeeding and disruption of hypothalamic insulin - signalling. other indirect mechanisms (in the presence of obesity) include hyperinsulinemia, increased leptin and nonesterified fatty acids (nefas) release from excess visceral fat, and reduced baroreceptor sensitivity and activation of the hypothalamic - pituitary - adrenal axis. the proposed mechanisms for sympathetic overactivity as a distinct consequence of metabolic dysfunction are discussed. animal and human studies clearly show that over- and underfeeding can modulate sympathetic nerve activity [3739 ]. it has been proposed that chronic sympathetic activity in obesity is an adaptive physiological response used to stimulate thermogenesis and stabilize body weight during periods of overeating. the notion that the autonomic nervous system acts to oppose weight change is supported by evidence from both normal weight and obese subjects that show modest weight gain is associated with an increase in sympathetic nerve activity and decrease in parasympathetic activity. chronic centrally mediated thermogenesis as a consequence of overfeeding does, however, come at the expense of sustained beta - adrenergic activation in the peripheral vasculature and kidneys which can precipitate a secondary rise in blood pressure via sodium retention and impaired pressure - natriuresis leading to obesity - related hypertension. insulin serves to reduce endogenous glucose production by directly inhibiting hepatic glycogenolysis or indirectly through the inhibition of lipolysis in adipose tissue, pancreatic glucagon production, or stimulation of insulin - dependent signalling pathways in the hypothalamus. there are several lines of evidence to suggest that insulin exerts sympathoexcitatory effects via actions in the central nervous system. indeed although insulin is not produced in significant amounts within the central nervous system, circulating insulin can gain access via saturable transport - mediated uptake across the blood - brain barrier [43, 44 ]. recently identified the arcuate nucleus in the hypothalamus as one of the critical sites where insulin acts to increase sympathetic nerve activity and the sympathetic baroreflex. it is postulated that the sympathoexcitatory effects of insulin result from suppressed inhibition of neuropeptide y (npy) neurons that project from the arcuate nucleus to the paraventricular nucleus. following a meal, centrally acting insulin exerts anorexigenic effects in the hypothalamus via stimulation of proopiomelanocortin (pomc) and inhibition of nyp neurons. however, during chronic overfeeding, npy gene expression in the arcuate nucleus is paradoxically elevated as a consequence of hyperinsulinemia - mediated alterations in insulin receptor and insulin - signalling pathways potentiating central insulin resistance. activation of npy neurons promotes potent orexigenic effects via increased sympathetic outflow to the liver resulting in hepatic insulin resistance and increased endogenous glucose production. in animals, intracerebroventricular administration of npy is shown to promote hyperphagia, hyperinsulinemia, insulin resistance, and obesity [4749 ]. in humans, systemic but not local infusion of insulin stimulates an increase in sympathetic nerve activity supporting the notion that insulin 's sympathoexcitatory effects are not mediated by a local mechanism. of note, the sympathetic response elicited by insulin in humans is far more heterogeneous than in animals. in healthy young adults, insulin infusion during constant plasma glucose concentration is shown to cause regionalised elevations in sympathetic nerve activity to skeletal muscle but not the kidneys. furthermore the effect of acute hyperinsulinemia on muscle sympathetic nerve activity is more pronounced in lean adults compared to obese. in hyperinsulinemic obese individuals with chronically elevated levels of circulating insulin, the normal central effects of insulin are blunted leading to increased endogenous glucose production (via activation of sympathetic outflow to the liver) and sustained sympathetic activation via the insulin feedback loop. given the observation that hyperinsulinemia causes regionalised sympathetic activity only (specifically to skeletal muscle) and subjects with obesity and hypertension are shown to have elevated renal noradrenaline spillover to plasma it is questionable whether hyperinsulinemia is the main mediator of central sympathetic overactivity observed in the mets. visceral white adipose tissue is a highly metabolic organ that not only accompanies but antedates other components of the mets including insulin resistance, hypertension, hyperglycemia, and inflammation. among the various indices of obesity, it is abdominal visceral obesity that is shown to be the strongest determinant of muscle sympathetic nerve activity in adults, with sympathetic nerve firing 55% greater in males with abdominal visceral obesity compared to men with subcutaneous obesity [24, 53 ]. animal and human data show certain adipokines expressed by white adipose tissue, namely, leptin and nonesterified fatty acids, can contribute indirectly towards sympathetic nerve activity. leptin is present in serum concentrations directly proportionate to adipose tissue mass and is shown to be elevated in human obesity. it can act directly on skeletal muscle to impair glucose transporter-4 (glut-4) translocation and induce hyperinsulinemia resulting in coactivation of the sympathetic nervous system. alternatively, leptin can act centrally in several brain regions (primarily the hypothalamus and brainstem) that control multiple metabolic functions via melanocortin - system - dependent pathways to increase sympathetic activity. very recent data suggests that leptin also acts at the level of the nucleus of the solitary tract to alter neurons involved in baroreflex sensitivity. the main central effect of leptin is a reduction in appetite and increase in energy expenditure via sympathetic activation. it has been postulated that, in obesity, neurons located in the ventromedial hypothalamic nucleus that express leptin receptors become desensitized to chronically elevated levels of leptin (hyperleptinemia) suppressing its anorexigenic effects while selectively preserving sympathoexcitation (known as selective leptin resistance) [57, 58 ]. animal models of obesity support this with leptin resistance in the hypothalamus shown to decrease satiety but preserve sympathetic activity. while acute administration of leptin elicits a marked rise in muscle sympathetic activity in healthy lean men there is some doubt as to whether leptin is the principal driver of chronic sympathetic activation in the mets. compared to nonobese adults, adults with high levels of subcutaneous fat who display 2 - 3-fold higher plasma leptin levels do not exhibit increased muscle sympathetic nerve activity. furthermore longitudinal studies in young japanese adults reveal elevations in plasma noradrenaline levels precede both weight gain and increases in plasma leptin levels [29, 61 ] suggesting hyperleptinemia is ancillary to sympathetic stimulation associated with obesity. several studies in human show a positive correlation between circulating plasma nonesterified fatty acids (nefas), obesity, and insulin resistance. as with leptin, nefas are able to act locally in peripheral tissue to disrupt insulin signaling and impair glucose uptake or can act centrally. although it is unclear how nefas activate central sympathetic nerve activity, the infusion of nefa is shown to increase muscle sympathetic activity in lean healthy adults. there is also evidence that central sympathetic activation contributes to the increased alpha - adrenoceptor mediated pressor sensitivity observed with acute elevations in nefa. despite evidence to support a possible interaction between nefa and the sympathetic nervous system, others have shown no change in whole - body and renal noradrenaline spillover during nefa infusion highlighting the need for further research in this area. stress - induced elevations in glucocorticoids are associated with profound metabolic abnormalities including insulin resistance, glucose intolerance, dyslipidemia, increased central adiposity, and hypertension suggesting that chronic stress may in part contribute to the development of the mets. findings from a nested, case - control study of the whitehall ii study show both the hypothalamic - pituitary - adrenal hpa axis and sympathetic nerve activity are elevated in mets subjects providing a possible causal link between chronic stress and sympathetic activation. experimental findings from animal and human studies lend further support to the coactivation of these brain centres during metabolic abnormalities, that is, obesity - induced hyperinsulinemia. direct intracerebroventricular administration of corticotropin - releasing hormone (crh) in primates is shown to promote sympathoexcitatory effects that lead to an increase in plasma noradrenaline levels, hyperglycemia, and elevations in blood pressure. importantly, these effects are only attenuated following administration of a ganglionic blocker not a crh antagonist suggesting hyperglycemia associated with hpa axis activation is secondary to central sympathetic activation. in obese adults with increased sympathetic nerve activity, chronic administration of dexamethasone is shown to attenuate elevations in both plasma cortisol and muscle sympathetic nerve activity but not in lean adults. the arterial baroreflex is designed to buffer beat - to - beat fluctuations in arterial blood pressure. the sympathoinhibition and sympathoexcitatory effects induced by baroreceptor stimulation and deactivation, respectively, are impaired. arterial baroreflex activity is also blunted in patients with visceral obesity, hypertension, insulin resistance, and early onset diabetes suggesting baroreflex impairment may play a causal role in the sympathoexcitatory state observed in the mets. baroreceptors located in the carotid sinus and aortic arch are highly sensitive to stretch in the vessel wall and it has been postulated that reduced arterial distensibility can blunt baroreceptor discharge during increased arterial pressure resulting in chronic stimulation of efferent sympathetic outflow to the periphery. in support of a primary role of the sympathetic nervous system in metabolic abnormalities that cluster to form the mets, several prospective studies clearly show that elevated noradrenaline levels can precede clinical manifestations of obesity and hypertension. in an elegant study of japanese ship builders, masuo. showed increased levels of circulating plasma noradrenaline independently predicted weight gain, elevated insulin levels, and blood pressure elevation during a 10-year follow - up period [29, 61 ]. similar results over a 20-year follow - up period have been observed for weight gain in norwegian men. first proposed that increased sympathetic activity was the primary defect leading to insulin resistance and weight gain in obese adults. increased sympathetic nerve activity is vital in the dissipation of energy following food consumption via activation of beta - receptors and it is proposed that chronic sympathetic nerve activity can potentiate weight gain leading to obesity as a consequence of diminished sensitivity of beta - adrenoceptors. in vitro and in vivo studies clearly show that prolonged adrenergic stimulation results in desensitization of beta - receptor mediated responses [75, 76 ]. downregulation of beta - adrenoceptors leading to a blunted thermogenic response to food can potentiate insulin resistance and perpetuate the negative feedback cycle between insulin governing sympathetic outflows. evidence in support of weight gain being directly related to decreased beta - adrenergic responsiveness comes from studies that show that both short - term and chronic [78, 79 ] pharmacological blockade of beta - receptors leads to an increase in body weight. due to the complex interactions between the sympathetic nervous system, hyperglycemia, hyperinsulinemia, metabolism, and insulin resistance it is difficult to define the primary insult that leads to metabolic dysfunction. as alluded to above, there is evidence in support of hyperinsulinemia promoting sympathetic nerve activity. it has also been proposed that insulin resistance is a secondary phenomenon precipitated by an increase in sympathetic tone [20, 80 ]. evidence that sympathetic overactivity precedes the development of insulin resistance and prediabetes is supported by findings from a study of young norwegian males where elevation in plasma norepinephrine during a cold pressor test was shown to predict hyperglycemia and impaired insulin sensitivity (as measured by homa - ir index) at 18-year follow - up. increased sympathetic outflow to skeletal muscle plays an important role in glucose metabolism primarily through eliciting reductions in skeletal muscle blood flow. indeed, an acute increase in sympathetic nerve activity has been shown to cause insulin resistance in the forearm of healthy adults. alpha - adrenergic vasoconstriction resulting from chronic sympathetic activity can blunt postprandial increases in skeletal muscle blood flow impairing glucose uptake and stimulating additional insulin production by the pancreas leading to insulin resistance. in support of elevated sympathetic nerve activity promoting insulin resistance through peripheral vasoconstriction, administration of peripherally acting vasoactive agents has been shown to improve insulin sensitivity in obese hypertensive patients [83, 84 ]. given the important role sympathetic overactivity plays in metabolic abnormalities, inhibition of the sympathetic nervous system represents a logical and attractive therapeutic approach to treat the mets and potentially reduce overall diabetes and cvd risk. it is possible to reduce central sympathetic activity through lifestyle modification, namely, energy - restricted diets and physical training [6, 10, 85 ] although recent evidence suggests pharmacological and device - based interventions targeting central sympathetic outflow are also likely to be of benefit. evidence from the diabetes prevention program and oslo diet and exercise study clearly demonstrate the important metabolic benefits that can be derived from intensive lifestyle interventions [86, 87 ]. whilst exercise training is shown to improve sympathoinhibition (preferentially to the kidneys) and potentiate baroreflex sensitivity [25, 89 ] there is strong evidence to suggest that weight loss, in particular abdominal fat loss, is the most important determinant of sympathetic neural adaption to improve hemodynamic and metabolic parameters in adults with the mets. in an elegant study by straznicky., middle - aged subjects with the mets who lost 9% of their body weight after undergoing a 12-week hypocaloric diet were shown to have significantly improved resting muscle sympathetic nerve activity, whole - body noradrenaline spillover, and whole - body insulin sensitivity. the addition of moderate - intensity physical activity may help augment the improvements observed in sympathoinhibition and metabolic outcomes. pharmacological inhibition of sympathetic nerve activity to achieve sustained weight loss and improvements in insulin sensitivity, glucose tolerance, dyslipidemia, and hypertension is currently under intense investigation. therapies targeting central sympathetic outflow, in particular, are thought to offer the greatest benefits as they may not only improve metabolic control but also help to regress end - organ damage. imidazoline i1 agonists act centrally at the level of the rostral ventrolateral medulla to inhibit sympathetic drive. moxonidine is a second - generation imidazoline i1 agonist for the treatment of mild - to - moderate hypertension via inhibition of central sympathetic outflow. moxonidine is also shown to exert beneficial effects on a diverse range of metabolic parameters including improvements in indices of glycaemic control, insulin sensitivity, dyslipidemia, and inflammation [9093 ]. it also promotes weight loss by lowering leptin plasma levels in obese patients and has been associated with improved renal function [95, 96 ], endothelial homeostatic mechanisms, and a reduction in left ventricular hypertrophy. the capacity of moxonidine to improve several metabolic measures in addition to its effect on blood pressure makes it an attractive therapeutic option for the treatment of mets and to reduce overall cvd risk. in a recent large, multinational, open - label 6-month uncontrolled observational study daily moxonidine use (either as a monotherapy or as an adjunct therapy) was shown to not only reduce blood pressure but also lower the cvd risk profile of mets patients. this was achieved by improving, albeit modestly, several metabolic indices ranging from weight reduction of 2.1 kg, decrease of 0.6 mmol / l in triglycerides, and decline of 5 beats / minute in heart rate. larger studies with longer duration of follow - up are needed to confirm these promising findings. catheter - based renal denervation has emerged as a safe and effective therapy to lower central sympathetic nerve activity. indeed, several studies show renal denervation lowers (by 50%) renal sympathetic nerve activity (as assessed by renal noradrenaline spillover), whole - body sympathetic nerve activity, and muscle sympathetic nerve activity. the therapy, which involves selective radiofrequency ablation of efferent and afferent sympathetic nerves in the renal artery lumen, is currently only recommended for use in patients with resistant hypertension to lower uncontrolled blood pressure. most recently, mahfoud. reported for 37 drug - resistant hypertensive patients who underwent renal denervation, improvements in systolic and diastolic blood pressure were accompanied by significant reductions in fasting plasma glucose, insulin, and c - peptide as well as 2-hour postload glucose levels at 3-month follow - up. insulin sensitivity as measured by the homa - ir and is quicki index measures was also improved at 6-month follow - up. these beneficial metabolic alterations were not observed in the control group (n = 13) who continued their usual medication regime. further support of the beneficial effects on glucose metabolism following renal denervation has been reported in patients with polycystic ovary syndrome and obstructive sleep apnea, two conditions that are characterised by multiple metabolic disturbances. in women with polycystic ovary syndrome, renal denervation was shown to lower fasting plasma glucose, improve insulin sensitivity (assessed by euglycaemic hyperinsulinemic clamp), and reduce both muscle sympathetic nerve activity and whole - body noradrenaline spillover at 3-month follow - up. importantly these metabolic effects were shown to occur in the absence of any changes in body weight. in a small study of 10 patients with obstructive sleep apnea, renal denervation was associated with improved 2-hour glucose levels during an oral glucose tolerance test and reduced hba1c levels at 6 months following renal denervation. while acute sympathetic activation may be desirable under certain circumstances, it is clear that chronic stimulation of the sympathetic nervous system has the potential to augment risk for the mets through the development of obesity, hyperglycaemia, insulin resistance, and hypertension. while the exact mechanisms are yet to be fully elucidated, several lines of evidence suggest that sympathetic nervous system overactivity is important both in the initiation and the maintenance of metabolic abnormalities commonly seen in the mets. latest findings from pharmaceutical and device - based clinical trials are encouraging for targeting central sympathetic activity to improve metabolic control in patients with the mets who are at heightened risk for diabetes and cardiovascular disease. from a research perspective, studies are needed to ascertain whether these latest therapies can ameliorate metabolic disease and attenuate future end - organ damage commonly associated with chronic sympathetic nerve activity.	sympathetic tone is well recognised as being implicit in cardiovascular control. it is less readily acknowledged that activation of the sympathetic nervous system is integral in energy homeostasis and can exert profound metabolic effects. accumulating data from animal and human studies suggest that central sympathetic overactivity plays a pivotal role in the aetiology and complications of several metabolic conditions that can cluster to form the metabolic syndrome (mets). given the known augmented risk for type 2 diabetes, cardiovascular disease, and premature mortality associated with the mets understanding the complex pathways underlying the metabolic derangements involved has become a priority. many factors have been proposed to contribute to increased sympathetic nerve activity in metabolic abnormalities including obesity, impaired baroreflex sensitivity, hyperinsulinemia, and elevated adipokine levels. furthermore there is mounting evidence to suggest that chronic sympathetic overactivity can potentiate two of the key metabolic alterations of the mets, central obesity and insulin resistance. this review will discuss the regulatory role of the sympathetic nervous system in metabolic control and the proposed pathophysiology linking sympathetic overactivity to metabolic abnormalities. pharmacological and device - based approaches that target central sympathetic drive will also be discussed as possible therapeutic options to improve metabolic control in at - risk patient cohorts.
it is well known that free radicals, including reactive oxygen species (ros) such as superoxide (o2) or hydroxyl radical (ho), contribute to the development of several diseases by causing oxidative stress. oxidative stress induced by ros occurs when the production of ros exceeds the capacity of the cell to detoxify these potentially injurious oxidants using endogenous antioxidant defense systems ; however, where an excessive amount of ros is produced or defense mechanisms are impaired, oxidative stress leading to events such as lipid peroxidation may occur. one well - accepted example for the mechanism of ros - induced vascular disease, such as hypertension, is that oxidative stress caused by excess o2 scavenging endothelial nitric oxide (no ; a vascular smooth muscle relaxant) could contribute to increased vascular smooth muscle contraction and hence elevated total peripheral resistance. these phenomena would also appear to be important in contributing to several pathological conditions in the central nervous system (cns), including stroke. therefore, ros - induced oxidative stress has been implicated as a potential contributor to the pathogenesis of the ischemia - reperfusion cns injury characteristic of hypertension and stroke. electron spin resonance (esr) has been recognized as one of the most powerful techniques for the detection of ros in the form of free radicals, in biological tissues and cells. it has been developed an esr - based technique to determine the effects of reactions induced by ros in biological systems in vitro and in vivo with an emphasis on a rodent disease model. regarding in vitro esr applications, spin trapping technique is well known as esr spin adduct to detect ros, quantifying spin concentration in experimental systems. otherwise, for in vivo esr applications, nitroxyl radicals are very useful as exogenous spin probes to measure free radical distribution, oxygen concentration, and redox metabolism in biological systems. it has been suggested that the blood brain barrier (bbb)-permeable molecule 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (mc - proxyl) is a suitable spin probe for the study of free radical reactions induced by ros in the brains of small animal models using in vivo esr detection. using a combination of these esr methods collectively focused on animal models of disease, recent results show that oxidative stress plays a key role in the vascular injury that occurs in hypertension and stroke. the spontaneously hypertensive rat (shr) or stroke - prone shr (shrsp), models of essential hypertension or stroke, respectively, exhibit several characteristics of the increased oxidative stress induced by ros. this review will focus on oxidative stress induced by ros, which have a critical role in the pathogenesis of vascular disease in the brain, in hypertension, or in stroke, using the rodent model. thus, we demonstrate in this review that results obtained from our laboratory by in vitro or in vivo esr techniques could serve as a useful basis for evaluating oxidative stress induced by ros in the brain of a rodent disease model, such as shr or shrsp. furthermore, esr techniques may be applicable to the assessment of antioxidant properties of drugs or food factors used for clinical treatment of human ros - induced conditions following their use for investigation into rodent disease models. the only technique that can see free radicals directly and specifically is esr because it detects the presence of unpaired electrons. however, esr detects only fairly unreactive radicals, since reactive ones do not accumulate to sufficiently high levels to be measured. one solution to this problem is to add spin traps or spin probes, agents that intercept reactive radicals, reacting with them to form a stable radical that can be detected by esr. thus, esr has been recognized as one of the most powerful techniques for the detection of ros in the form of free radicals, in biological tissues and cells. it has been developed an esr - based technique to determine the effects of ros - mediated reactions in biological experiments in vitro and in vivo, with an emphasis on rodent disease models such as the shr and shrsp models. regarding in vitro esr applications, spin trapping technique is a well known technique for detecting and quantifying ros concentrations in experimental systems. we previously reported various effects of ros such as ho, singlet oxygen (o2), hydrogen peroxide (h2o2), and o2 in experimental biological systems using the esr spin trapping technique. nitroxyl radicals are very useful as exogenous spin probes for the measurement of the free radical distribution in biological systems, especially stable radicals such as nitroxyl radical 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl - proxyl) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (hydroxy - tempo). these are often used as spin probes for in vivo esr, operating in the lower - frequency microwave bands (1 ghz), the so - called l - band, where the dielectric losses of biological systems are lower. during the last decade, significant advances in l - band and in vivo esr techniques have provided useful information on oxidative stress in biological systems. the signal decay rate of the nitroxyl radical gives evidence of oxidative stress induced by ros and changes of redox status in biological systems. we have used l - band esr to characterize the higher degrees of oxidative stress in the shrsp and shr than in the wky brain, and the lower extent of oxidative stress in the shr than in the shrsp brain. significant advances in the field of esr imaging in recent years have now made it possible to visualize the distribution and metabolism of free radicals, and the degree of tissue oxygenation in vivo. in vivo esr spectroscopy and esr imaging are useful for investigation of the redox status of living organisms non - invasively. esr imaging is particularly useful for determining the in vivo spatial distribution of free radicals in animals. it has been suggested that bbb - permeable mc - proxyl is a suitable spin probe for the study of free radical reactions in the brain by in vivo esr. since the permeability of the bbb to a compound is dependent on its lipophilicity and molecular weight, the partition coefficients between 1-octanol and water of various nitroxyl compounds have been measured (see miura.). the partition coefficient values of carbamoyl - proxyl (0.87) and hydroxy - tempo (4.83) were consistent with those reported by fuchs. mc - proxyl was more lipophilic (partition coefficient = 8.70) than carbamoyl - proxyl and other spin probes, implying high permeability of the bbb to this compound. our study also demonstrated that relatively large amounts of mc - proxyl were distributed to the brain compared with the bbb - impermeable carbamoyl - proxyl. this was observed using esr imaging for 3d or esr - computed tomography (ct) of the head regions of mice (fig. the results of the several studies mentioned above indicate that the in vivo esr / nitroxyl spin probe techniques employed herein could be a powerful tool to detect free radicals selectively and to monitor free radical reactions in vivo. quantitative esr analysis using mc - proxyl has the potential to be useful for understanding redox status under conditions of oxidative stress in the rodent brain. oxidative stress occurs when the production of ros exceeds the capacity of the cell to detoxify these potentially injurious oxidants using endogenous antioxidant defense systems. conditions associated with oxidative stress include ischemia / reperfusion, hypercholesterolemia, diabetes, and hypertension. the adhesion of circulating blood cells (leukocytes, platelets) to vascular endothelium is a key element in the pro - inflammatory and prothrombogenic phenotype assumed by the vasculature in these and other disease states that are associated with oxidative stress. there is a growing body of evidence that links the blood cell - endothelial cell interactions in these conditions to the enhanced production of ros. it is well known that the mechanism of vascular disease, such as hypertension, is due to oxidative stress caused by excess o2 scavenging endothelial no ; this can contribute to increased vascular smooth muscle contraction and hence elevated total peripheral resistance. we have also demonstrated that the biphasic concentration - dependent regulation of polymorphonuclear leukocyte (pmn)-induced o2 generation by no - derived peroxynitrite (onoo) may be of critical importance in regulating processes of inflammation such as ischemia - reperfusion (fig. 2). these small critical molecules exert opposing effects on vascular tone and chemically react with each other ; this invalidates their individual effects and leads to the formation of onoo (fig. 2). because of the apparent importance of ros (and in particular o2) in vascular disease, many sources of o2 have been investigated, including xanthine oxidase, nicotinamide adenine dinucleotide phosphate (nadph) oxidase, and leakage from mitochondria (fig. 2). it has demonstrated that the increased o2 production in endothelial cells as well as in vascular smooth muscle cells occurs via a membrane - associated nad(p)h oxidase (fig. 2). all cell types in the vascular wall produce ros derived from o2-generating protein complexes similar to the nadph oxidase expressed in pmns. specific features of the vascular enzymes include constitutive and inducible activities, substrate specificity, and intracellular o2 production. interestingly, this oxidase is activated upon stimulation with angiotensin ii (fig. 2). this suggests that an activated circulating and/or local renin - angiotensin system, and subsequent increased vascular o2 production, are common risk factors for hypertension (fig. furthermore, our laboratory obtained the first evidence that ho and singlet oxygen (o2) directly induced vascular contraction. taken together, the mechanism of development of hypertension may involve increases in ros generation in the vascular system (fig. 2). stroke initiates a complex cascade of metabolic events, several of which involve the generation of ros. the vascular system is the first target of ros generated in several pathological process, and vascular damage is a prominent feature of embolic stroke. we have already confirmed the generation of ros, mainly o2, via the nadph oxidase - derived oxidant burst of activated pmns, or the xanthine - xanthine oxidase system (fig. 2). furthermore, there is evidence that ros may play a critical role in vascular reactivity in cerebral arteries ; o2 may then escape into the extracellular space via an anion channel, after which o2 rapidly dismutates to h2o2 through catalytic reaction with superoxide dismutase. potent ros, ho, can be formed by the haber - weiss reaction from o2 and h2o2. however, this reaction is considered too slow to compete with the dismutation reaction. ho can be generated more efficiently via the haber - weiss reaction, if ferrous iron is present, via a reaction known as the biological fenton reaction. exposure of cellular components of vascular smooth muscle to exogenous ros can lead to cellular dysfunction and necrosis. thus, ros released acutely in large amounts have been traditionally implicated in the cell death associated with vascular ischemia - reperfusion injury. therefore, ros are crucial factors in the pathogenesis of vascular incompetence following vascular damage, such as that due to stroke. activation of the nadph oxidase - derived oxidant burst of pmns is of more critical importance in stroke than in hypertension because a large amount of o2 could be generated by nadph oxidase expressed in pmns (fig. 2). further pmn - derived o2 can be scavenged by no, with the formation of the potent toxic onoo (fig. 2). thus, it should be noted that severe hypertension and cerebrovascular diseases induced by excess ros generation can lead to stroke. therefore, we need to know the differences in the level of oxidative stress induced by ros in hypertension and stroke. it is likely that this information will be useful in understanding mechanisms of hypertension or stroke in relation to oxidative stress induced by ros. the development of genetic models for research on hypertension and stroke (shr and shrsp) has contributed to the ability to predict and prevent these conditions. cerebral ischemia and reperfusion cause delayed neuronal death in rodents such as mongolian gerbils and shrsp, both of which have been used as experimental stroke models. the shr, a model of essential hypertension, has several characteristics of increased oxidative stress (fig. 2). there is significant in vitro evidence indicating that o2 contributes to increased systemic vascular tone in shr. it has also been shown by in vivo fluorescence microscopy that blood vessels of shr generate excessive amounts of o2. ros generation occurs after reperfusion and this free radical - induced oxidative stress can greatly damage neurons in the shrsp. oxidative stress induced by excessive generation of o2 in shrsp tissues leads to increased hypertensive responses, which may be relevant to the pathophysiology of stroke (fig. 2). this would therefore be an important mechanism of oxidative stress for production of excessive ros, mainly o2, in animal models such as shr or shrsp (fig. 2). in vivo l - band esr and l - band esr imaging methods have also been employed in brains from normotensive rats (wistar - kyoto rat (wky)), shr, and shrsp using mc - proxyl. we obtained pharmacokinetic profiles of mc - proxyl esr signals in the shr and shrsp brain. these can be divided into phase i and phase ii, according to a two - compartment model of distribution, using in vivo l - band esr (fig. nitroxyl spin probes are administered systemically and are eliminated principally by a combination of excretion via the kidney and reduction to the hydroxyl amines. the decay of the nitroxyl spin probe, mc - proxyl, which is reflected in esr signal intensity in the head region, appears to follow the usual pharmacokinetic pattern for excretion of drugs : those that tend to stay in the vascular system are excreted very rapidly (phase i) due to reduction by ascorbate, while lipophilic nitroxyl spin probes, such as mc - proxyl, show much slower excretion via the kidney (phase ii) (fig.. the decay rate constants for mc - proxyl in phase i (k1) and phase ii (k2) in the brain were faster in shr and shrsp than in wky. furthermore, the phase i decay rates (k1) in shrsp were more rapid than in shr, while no significant difference was observed in phase ii decay rates (k2) between shrsp and shr (fig. these results suggest that the higher oxidative stress in shrsp brain occurred in the cerebral vascular system rather than in the tissue responsible for degrading the mc - proxyl esr signal in the brain. taken together, our data indicated that the level of oxidative stress in the cerebrovascular system in rodent brain was in the following descending order : shrsp > shr > wky (fig. 3 and 6), which is the same descending order as for blood pressure in these animals. it has already been demonstrated that no differences exist between shr and wky with respect to blood flow in the heart, brain, lungs, spleen, and adrenal. in spite of the large differences in arterial pressure, blood flow in the brain thus, the pathophysiology of hypertension and therefore stroke could involve increases in the level of oxidative stress by ros generation in the cerebrovascular system (fig. 3 and 6). the most convincing evidence that oxidative stress induced by ros generation is involved in brain disease such as stroke is the repeated observation that compounds that inhibit lipid peroxidation, or scavenge ros, can prevent post - traumatic pathophysiological changes and promote functional recovery and survival in experimental studies. nevertheless, the significance of ros and lipid peroxidation ultimately depends on whether it can be demonstrated that early application of an effective ros scavenger can promote survival and neurological recovery after cns injury and stroke in humans. it is well known that ros scavengers may decrease edema and tissue damage in stroke. thus, the neuroprotective properties of anti - stroke agents could be associated with their antioxidant properties, indicating ros scavenging activity. however, there is very little direct evidence of their antioxidant properties, specifically due to a lack of techniques for detecting ros and for the assessment of oxidative stress levels in vivo, particularly in a suitable stroke animal model. therefore, we need further studies using esr techniques, both in vitro and in vivo, aimed at characterizing antioxidant properties of neuroprotective agents. propofol anesthesia has been associated with lower intracranial pressure and cerebral swelling than volatile anesthesia in brain tumor patients undergoing craniotomy. the potential neuroprotective effect of propofol may be mediated by its antioxidant properties, which have been shown to play a role in apoptosis, ischemia - reperfusion injury and inflammatory - induced neuronal damage, due to reduction of lipid peroxidation via the generation of ros. our present results provide the first direct evidence of the antioxidant properties of propofol and a vehicle such as medium - chain triglyceride / long - chain triglyceride (mct / lct) using in vitro esr spin tapping technique. interestingly, we found that the effects of the vehicle are most probably due to scavenging ho from the biological fenton s reaction in tissues such as the brain. secondly, we confirmed that oxidative stress in the brain of shrsps was higher than that in wkys anesthetized with pentobarbital (fig. when propofol mct / lct was used as an anesthetic, rather than pentobarbital, the level of oxidative stress in shrsp then became similar to that seen in wky (fig. 4b), suggesting that propofol mct / lct reduced shrsp - induced oxidative stress in the brain. consequently, propofol mct / lct could be particularly useful in adjusting levels of anesthesia in cases of ros - induced brain disease. crocetin is a natural carotenoid compound found in the stigmas of saffron (crocus sativus l.) and the fruits of gardenia jasminoides ellis. this yellow compound has been used as an important spice and natural food colorant in various parts of the world. in addition, saffron and gardenia fruits have been used as traditional medicine and crocetin is one of the major active compounds of these herbal medicines. in the present study, we used the esr technique to investigate the ros scavenging effect of food factors such as crocetin and the decay rate constant of mc - proxyl in the isolated brain of the shrsps. the results showed that crocetin to shrsps was capable of reducing ros - mediated oxidative stress in the brain due to a direct antioxidant effect, indicating that esr technique could be useful for the assessment of the antioxidant property of food factors such as crocetin. a better knowledge of clinical and laboratory markers of functional outcome would result in a more individualized and possibly improved approach to management of patients with oxidative stress - induced diseases such as hypertension or stroke. it is well accepted that infarct size as detected in neuroimaging studies constitutes a strong predictor of clinical outcome. however, ct scan or brain magnetic resonance imaging (mri) information concerning the extent of a cerebral infarction is usually available too late after clinical onset to be of help in the decision - making phase. the predictive value of neuroimaging is then limited to the long - term phase of stroke. as a result, prediction of stroke outcome basically relies on clinical findings. as previously mentioned, we have now developed an in vivo esr imaging system with high - quality esr - ct images or reconstructed 3d images by using mc - proxyl in a rodent model (fig. it should be noted that such a clinical indicator of acute stroke provides little information in relation to the prevention of stroke. further advances in the instrumentation used for esr imaging and in the development of optimized nontoxic spin probes will make esr technology one of the most novel diagnostic tools for acute stroke or clinical predictors for prevention of stroke in the future. we have already indicated that our esr assessment can characterize the degree of oxidative stress in the shr model of hypertension and the shrsp model of stroke using by esr imaging system (fig. it is likely that the level of oxidative stress using esr assessment may give useful information on pathological developments in the progression from hypertension to stroke. another limitation is the lack of measures of antioxidants and antioxidant status within recently reported studies. since we have evaluated the degree of oxidative stress in the brain of shr and shrsp as mentioned above, this means that we have now effectively established novel in vivo esr methods for assessment of antioxidant properties of drugs such as propofol mct / lct or food factors such as crocetin using shr or shrsp. in conclusion, we have demonstrated that esr methods using spin trap or spin probe will be useful for understanding redox status under conditions of oxidative stress in the shr or shrsp brain. we have used esr imaging and noninvasive l - band esr to characterize the higher degrees of oxidative stress in the shrsp or shr than in the wky brain, and the lower extent of such stress in the shr than in the shrsp brain. indeed, we may be able to confirm propofol mct / lct as neuroprotective anesthesia or crocetin as an antioxidant food factor against human stroke after screening for antioxidant properties in stroke models such as shrsp. thus, our esr biomedical application suggests that it could be used to assess antioxidant effects on oxidative stress in the brain using the shr and shrsp animal models of disease. we hope that further advances in the instrumentation used for esr imaging and the development of optimized nontoxic spin probes will make this technology even more promising for novel clinical prediction or noninvasive diagnosis of human stroke. after screening drugs or food factors for antioxidant property using in vitro or in vivo esr assessment, it will be possible to find and develop novel drugs or food factors with such properties for the prevention of stroke in the near future.	the pathophysiology of hypertension or stroke is associated with an excess of ros generation in the vascular system, and results in induction of various pathological cascades of cerebrovascular damage. we have demonstrated that electron spin resonance methods using a spin trap or spin probe will be useful for understanding redox status under conditions of oxidative stress in the spontaneously hypertensive rat or stroke - prone spontaneously hypertensive rat brain. we have used electron spin resonance imaging and noninvasive l - band electron spin resonance to characterize the higher degree of brain oxidative stress in the stroke - prone spontaneously hypertensive rat and spontaneously hypertensive rat than in the wistar - kyoto rat brain, and the lower extent of oxidative stress in the spontaneously hypertensive rat than in the stroke - prone spontaneously hypertensive rat brain. indeed, we may be able to confirm propofol medium - chain triglyceride / long - chain triglyceride (mct / lct) as neuroprotective anesthesia and crocetin as antioxidant food factor against human stroke after screening for antioxidant properties in stroke models such as stroke - prone spontaneously hypertensive rat. thus, our electron spin resonance biomedical application suggests that it could be used to assess antioxidant effects on oxidative stress in the brain using spontaneously hypertensive rat and stroke - prone spontaneously hypertensive rat. we hope that further advances in the instrumentation used for electron spin resonance imaging and the development of optimized nontoxic spin probes will make this technology even more promising for novel clinical prediction or noninvasive diagnosis of human stroke. after screening drugs or foods for antioxidant property using in vitro or in vivo electron spin resonance assessment, it will be possible to find and develop novel drugs or food factors with such properties for the prevention of stroke in the near future.
percutaneous cardiopulmonary support (pcps) provides hemodynamic support via the femoral artery and vein, using a closed artificial heart - lung system which is usually composed of a centrifugal pump and a membranous artificial lung.1)2) in terms of cardiac failure, the most common indications for pcps are post - cardiotomy (in which cardiopulmonary bypass can not be taken off the patient following cardiac surgery), post - heart transplant (usually due to primary graft failure) and severe cardiac failure due to almost any other cause (eg., decompensated cardiomyopathy, myocarditis, acute coronary syndrome with cardiogenic shock, profound cardiac depression due to drug overdose or sepsis). in terms of respiratory failure, the most common indications include adult respiratory distress syndrome, pneumonia, trauma, and primary graft failure following lung transplantation.2 - 4) in particular, pcps has proven to be a technique of value in high - risk coronary patients who undergo percutaneous coronary intervention (pci).5 - 9) however, there have been few studies on pcps - supported pci in korea. we combined the use of pcps and coronary stent revascularization in 19 patients between august 2005 and june 2009, at the yonsei cardiovascular center. pcps was used as an elective procedure in 10 patients who satisfied at least two of the selection criteria : left ventricular ejection fraction (lvef) < 35%, target vessel(s) supplying more than 50% of the viable myocardium, high risk surgical patients { old age, impending end stage of renal disease, chronic obstructive pulmonary disease, a history of coronary artery bypass graft surgery (cabg) }, and patients who refused cabg surgery. in the remaining 9 patients, pcps was used as an emergency procedure to stabilize and even resuscitate patients with acute myocardial infarction (ami) and cardiogenic shock, in order to attempt urgent pci. we compared the clinical characteristics, pcps and angiographic findings, and clinical outcomes of the survivors and non - survivors. the pcps system used in our study was the capiox emergency bypass system (terumo, inc., tokyo, japan), which is an all - in - one package of a heparin - coated membrane oxygenator that is preassembled with bypass circuits and a cone for the centrifugal pump. the pcps apparatus is ready for emergency use, by a well - trained team at our hospital, ready to prepare the patient for pump placement within minutes. for the percutaneous technique, the tip of the arterial cannula was advanced and positioned in the common iliac artery ; the tip of the venous catheter was placed at the junction of the right atrium and the superior vena cava along a rigid backup guidewire under the fluoroscopic guidance. the patients were fully heparinized with continuous injection at the rate of 3 mg / kg / min, to maintain an activated clotting time greater than 200 seconds. the centrifugal pump provided a non - pulsatile flow at a rate between 1 and 5 l / min. during the procedure then, both cannulas were clamped, and the pump was interrupted. at this point, one - to - one protamine neutralization of the circulating heparin was performed, allowing removal of the cannulas and prolonged groin compression. the data are expressed as meansstandard deviations for the continuous variables, and as absolute and relative frequencies for the categorical variables. student 's unpaired t - test was used to compare the continuous variables between the groups. event - free survival curves were constructed by the use of the kaplan - meier method. we combined the use of pcps and coronary stent revascularization in 19 patients between august 2005 and june 2009, at the yonsei cardiovascular center. pcps was used as an elective procedure in 10 patients who satisfied at least two of the selection criteria : left ventricular ejection fraction (lvef) < 35%, target vessel(s) supplying more than 50% of the viable myocardium, high risk surgical patients { old age, impending end stage of renal disease, chronic obstructive pulmonary disease, a history of coronary artery bypass graft surgery (cabg) }, and patients who refused cabg surgery. in the remaining 9 patients, pcps was used as an emergency procedure to stabilize and even resuscitate patients with acute myocardial infarction (ami) and cardiogenic shock, in order to attempt urgent pci. we compared the clinical characteristics, pcps and angiographic findings, and clinical outcomes of the survivors and non - survivors. the pcps system used in our study was the capiox emergency bypass system (terumo, inc., tokyo, japan), which is an all - in - one package of a heparin - coated membrane oxygenator that is preassembled with bypass circuits and a cone for the centrifugal pump. the pcps apparatus is ready for emergency use, by a well - trained team at our hospital, ready to prepare the patient for pump placement within minutes. for the percutaneous technique, the tip of the arterial cannula was advanced and positioned in the common iliac artery ; the tip of the venous catheter was placed at the junction of the right atrium and the superior vena cava along a rigid backup guidewire under the fluoroscopic guidance. the patients were fully heparinized with continuous injection at the rate of 3 mg / kg / min, to maintain an activated clotting time greater than 200 seconds. the centrifugal pump provided a non - pulsatile flow at a rate between 1 and 5 l / min. during the procedure then, both cannulas were clamped, and the pump was interrupted. at this point, one - to - one protamine neutralization of the circulating heparin was performed, allowing removal of the cannulas and prolonged groin compression. the data are expressed as meansstandard deviations for the continuous variables, and as absolute and relative frequencies for the categorical variables. student 's unpaired t - test was used to compare the continuous variables between the groups. event - free survival curves were constructed by the use of the kaplan - meier method. the demographics and pre - existing co - morbidities were similar between the survivors and non - survivors. unstable angina and non - st elevation myocardial infarction patients were more common among the survivors ; st elevation myocardial infarction (stemi) with cardiogenic shock was more prevalent in the non - survivor group compared to the survivors (75% vs. 27.3%, p=0.04). the cardiac biomarkers (creatine kinase - mb, n - terminal pro - b - type natriuretic peptide) were not significantly different between the survivors and non - survivors either (table 1). the elective pcps - supported pci was more frequently practiced among the survivors compared to the non - survivors (72.7% vs. 25%, p=0.04). all patients were weaned from pcps in the survivor group and 6 (75%) patients were weaned from pcps among the non - survivors. the median durations of pcps support were not significantly different between the survivors and non - survivors. there were no significant differences in culprit lesions between the survivors and non - survivors. more patients with multi - vessel disease were in the survivors than in the non - survivors (90.9% vs. 50%, p=0.046), however, a lower thrombolysis in myocardial infarction (timi) grade (0 - 1) in the culprit lesions before pci was detected among the non - survivors. there were no significant differences in the use of intra - aortic balloon pump (iabp) or in the number of stents between the survivors and non - survivors (table 2). patients who survived were discharged from the hospital and closely followed at the outpatient department. overall in - hospital mortality was 31.6% (6 of 19), of which 4 deaths were cardiac - related and 2 were not. the causes of non - cardiac - related deaths were sepsis associated with pneumonia and upper gastrointestinal bleeding (table 3). we also compared the survival rates of patients who underwent elective procedure and emergency procedure. in the analysis of the event free survival curve between elective procedure and emergency procedure, although we studied high - risk patients, there was no procedure - related mortality. the most common complications were hematomas near the catheter insertion site, which disappeared by the time of discharge from hospital. one patient who underwent emergency procedure had lower limb ischemia and gangrene, but survived without sequelae. the demographics and pre - existing co - morbidities were similar between the survivors and non - survivors. unstable angina and non - st elevation myocardial infarction patients were more common among the survivors ; st elevation myocardial infarction (stemi) with cardiogenic shock was more prevalent in the non - survivor group compared to the survivors (75% vs. 27.3%, p=0.04). the cardiac biomarkers (creatine kinase - mb, n - terminal pro - b - type natriuretic peptide) were not significantly different between the survivors and non - survivors either (table 1). the elective pcps - supported pci was more frequently practiced among the survivors compared to the non - survivors (72.7% vs. 25%, p=0.04). all patients were weaned from pcps in the survivor group and 6 (75%) patients were weaned from pcps among the non - survivors. the median durations of pcps support were not significantly different between the survivors and non - survivors. there were no significant differences in culprit lesions between the survivors and non - survivors. more patients with multi - vessel disease were in the survivors than in the non - survivors (90.9% vs. 50%, p=0.046), however, a lower thrombolysis in myocardial infarction (timi) grade (0 - 1) in the culprit lesions before pci was detected among the non - survivors. there were no significant differences in the use of intra - aortic balloon pump (iabp) or in the number of stents between the survivors and non - survivors (table 2). patients who survived were discharged from the hospital and closely followed at the outpatient department. overall in - hospital mortality was 31.6% (6 of 19), of which 4 deaths were cardiac - related and 2 were not. the causes of non - cardiac - related deaths were sepsis associated with pneumonia and upper gastrointestinal bleeding (table 3). we also compared the survival rates of patients who underwent elective procedure and emergency procedure. in the analysis of the event free survival curve between elective procedure and emergency procedure, although we studied high - risk patients, there was no procedure - related mortality. the most common complications were hematomas near the catheter insertion site, which disappeared by the time of discharge from hospital. one patient who underwent emergency procedure had lower limb ischemia and gangrene, but survived without sequelae. based on the results of previous studies the application of pcps - supported pci on high risk patients with severe lv dysfunction is accepted as helpful.5 - 9) pcps provides complete systemic hemodynamic support independent of intrinsic cardiac function and rhythm, and has been successfully used prophylactically in high - risk patients during pci. this preventative support allows for precise diagnostic and therapeutic occlusive maneuvers { e.g., intravascular ultrasound (ivus), kissing balloon of bifurcated lesions } while preserving systemic perfusion pressures, and preventing both circulatory collapse and malignant arrhythmia.10)11) in one study, although it was from a small number of patients, the authors recommended prophylactic cardiopulmonary bypass support in unstable patients undergoing angioplasty with ef < 25%, or in patients whose ef is low and in whom the only patent artery is the target of dilatation.12) even stable patients with ef < 20% may benefit from prophylactic cardiopulmonary bypass support.7) pcps may also be helpful when used as an emergency procedure to recover hemodynamic stability in patients with ami and cardiogenic shock. pcps markedly decreases myocardial oxygen consumption and work, creating favorable hemodynamic conditions to impede progression of myocardial infarction. complications such as ventricular arrhythmias frequently appear in cardiogenic shock and are also transient and reversible under pcps. once the patient is stabilized, a coronary angiogram can be safely performed, after which a planned reperfusion and stent revascularization can be accomplished.13 - 15) in 1995, ha.16) reported, for the first time, a successful case of pcps - supported pci on a high risk patient. in 2006, rhee.17) reported the effectiveness of pcps on patients with cardiac arrest or cardiogenic shock. we analyzed, for the first time in korea, the high - risk patients who underwent pcps - supported pci, being the elective case group and emergency case group. even though the patients in both groups had similar pre - existing co - morbidities and lower ef, the patient conditions were quite different. those in the emergency group were in shock status even after inotropic drug administration, while patients in the elective group were in a relatively stable condition (under general anesthesia, transfusion, etc.). for this reason, among the survivors, the number of patients who underwent elective pcps - supported pci was significantly higher than among the non - survivors. also the in - hospital mortality rate was lower in the elective case group. in this study, elective pcps - supported pci was applied on 10 patients exhibiting at least two of the following conditions : lvef < 35%, target vessel(s) supplying more than 50% of the viable myocardium, high risk surgical patients and those who refused coronary bypass surgery. pcps was weaned off from all patients without serious procedure - related complications and there was no procedure - related mortality. due to underdevelopment of vascular device and stenting technology in the past, procedure - related complications were common in high risk patients who were subjected to application of lv assist.18) in this study, the use of a more advanced vascular device helped reducing procedure - related complications. we could expect a more favorable prognosis with elective pcps - supported pci on high risk patients. in the case of emergency pcps - supported pci, urgent pci was done to stabilize in patients who showed signs of stemi with cardiogenic shock and cardiac arrest. pcps was weaned off quite successfully (in 78% patients) after its application. however, the prognosis was not favorable, resulting in 44.4% of cardiac related death and 55.6% of in - hospital mortality. according to the results of previously published data, ami with cardiogenic shock led to a 80% mortality rate, and iabp, though still applied to lower the mortality rate, resulted a relatively high mortality rate.13)19) in this study, we were unable to restore cardiogenic shock of 5 patients by applying iabp alone, thus need a pcps - support. in other studies pcps - supported pci in emergency cases was more successful than that with iabp support alone.18) three patients of this study survived after pcps application and are now under outpatient department follow - up. however, further study about the benefits of this procedure will be needed, considering the small number patients, and their conditions (vital sign, co - morbidities, etc.), in this study. our study has some limitations : first, this is a retrospective observational analysis of a relatively very small number of patients. second, in the elective case group, we have not yet demonstrated that this approach is necessary or superior to unsupported angioplasty or to iabp, or to coronary bypass surgery where feasible. a randomized trial of supported versus unsupported (with cardiopulmonary support on standby) angioplasty maybe warranted. third, in the emergency case group, we have applied iabp first in most of our cases, thus it is hard to conclude that the benefits came from pcps alone. our experience suggests that pcps may be helpful in high - risk patients being or to be treated with pci, especially in the elective cases. however, pcps - supported pci still bears considerable risks in the emergent cases. to this day, a clear guideline or indication criteria have not been established for pcps guided pci. more aggressive and our study has some limitations : first, this is a retrospective observational analysis of a relatively very small number of patients. second, in the elective case group, we have not yet demonstrated that this approach is necessary or superior to unsupported angioplasty or to iabp, or to coronary bypass surgery where feasible. a randomized trial of supported versus unsupported (with cardiopulmonary support on standby) angioplasty maybe warranted. third, in the emergency case group, we have applied iabp first in most of our cases, thus it is hard to conclude that the benefits came from pcps alone. our experience suggests that pcps may be helpful in high - risk patients being or to be treated with pci, especially in the elective cases. however, pcps - supported pci still bears considerable risks in the emergent cases. to this day, a clear guideline or indication criteria have not been established for pcps guided pci.	background and objectivespercutaneous cardiopulmonary support (pcps) has proven to be a valuable technique in high - risk coronary patients undergoing percutaneous coronary intervention (pci). however, there have been few studies on pci associated with pcps in korea. we summarized our experience with pcps - supported pci.subjects and methodswe retrospectively reviewed 19 patients with pcps - supported pci between august 2005 and june 2009. pcps was used as an elective procedure for 10 patients with at least two of the following conditions : left - ventricular ejection fraction < 35%, target vessel(s) supplying more than 50% of the viable myocardium, high risk surgical patients, and patients who refused coronary bypass surgery. in the remaining 9 patients pcps was used as an emergency procedure, to stabilize and even resuscitate patients with acute myocardial infarction and cardiogenic shock, in order to attempt urgent pci.resultsamong the 19 patients who were treated with pcps - supported pci, 11 (57.9%) survived and 8 (42.1%) patients did not. st elevation myocardial infarction with cardiogenic shock was more prevalent in the non - survivors than in the survivors (75% vs. 27.3%, p=0.04). the elective pcps - supported pci was practiced more frequently in the survivors than in the non - survivors (72.7% vs. 25%, p=0.04). in the analysis of the event - free survival curve between elective and emergency procedures, there was a significant difference in the survival rate (p=0.025). among the survivors there were more patients with multi - vessel disease, but a lower thrombolysis in myocardial infarction grade in the culprit lesions was detected in the non - survivors, before pci. although we studied high - risk patients, there was no procedure - related mortality.conclusionour experience suggests that pcps may be helpful in high risk patients treated with pci, especially in elective cases. more aggressive and larger scale studies of pcps should follow.
streptococcus minor was described in 2004 as a new species isolated from the tonsils of dogs, cats and cattle as well as from the intestinal tract of dogs. although staphylococci and streptococci are commonly involved genera in bite - infected wounds, no cases of human infection by s. minor have been reported in the literature. here a 51-year - old woman was admitted for a dog bite injury to her right hand. the patient s medical history revealed a urinary bladder cancer 3 years ago, currently in remission. she presented with inflammation, redness, heat and pus production at the bite wounds (fig. 1). clinically, the patient was afebrile (temperature 36.6c), and no signs of systemic infection were observed. biologic investigations revealed an inflammatory syndrome with a c - reactive protein value of 46 mg / l (normal value, < 5 mg / l) associated with a light leukocytosis of 10.880 cells/l (normal range, 4 to 10 10 cells/l). urgent surgery was proposed but was refused by the patient despite warnings of the risk of sepsis and necrosis. the wounds were washed with povidone iodine, and intravenous empiric antibiotic therapy was initiated (cefazolin 2 g / d). one set of peripheral blood cultures (including aerobic and anaerobic conditions) was performed, and no growth was observed after 5 days of incubation. aerobic culture from pus wounds after 48 hours on columbia agar (becton dickinson, franklin lakes, nj, usa) yielded a pure growth of unpigmented, regular, translucent and -hemolytic colonies of gram - positive streptococcus. identification as streptococcus minor was achieved using matrix - assisted laser desorption / ionization time - of - flight mass spectrometry (maldi - tof ms ; bruker daltonics, bremen, germany), with an excellent score of 2.428. sequencing of 16s rrna gene (96ga3730 xl ; thermo fisher scientific life sciences, waltham, ma, usa) confirmed the identification of s. minor with 100% of identity and demonstrated the usefulness of maldi - tof ms for identifying this organism. the antimicrobial testing showed susceptibility to penicillin, clindamycin, erythromycin and trimethoprim sulfamethoxazole. the patient left the hospital the same day and received 10 days of oral antimicrobial therapy with amoxicillin clavulanate acid at 850/125 mg three times a day. under this antibiotic regimen, five days later, patient examination revealed no clinical signs of infection or radiologic signs of bone injury. infections caused by s. minor are probably underestimated because of the organism s facultative anaerobic nature, requiring a co2-enhanced atmosphere, and the difficulty in identifying to the species level -hemolytic streptococci with current laboratory techniques. moreover, in contrast to other streptococci generally identified in zoonotic infection, such as streptococcus canis (group g) involved in several cases of septicaemia and one case of infective endocarditis,, s. minor does not react with lancefield groups a, c, d, f or g antisera. although the pathogenicity of this organism is still unknown, this case suggests that s. minor can lead to serious local infections. this study also demonstrates the usefulness of maldi - tof ms for identifying this organism. to our knowledge s. minor has never been isolated from humans but is part of the commensal flora from dogs, suggesting strong causality. unfortunately, in this case, the dog bite had occurred a while ago, and the dog was not available for further testing in order to compare the homology of both the patient s and dog s isolate strains and to confirm the origin and the pathogenicity of s. minor. further investigation on the oral dog flora and on s. minor s virulence factors is necessary to confirm its implication and elucidate its pathogenic mechanisms.	we report the first case of human infection caused by streptococcus minor in a 51-year - old immunocompetent woman admitted for dog bite injuries. at present, the role of streptococcus minor in bite wound infections is unknown. further studies on virulence factors are needed to elucidate its pathogenicity mechanisms.
overabundant food intake with chronic positive energy balance leads to obesity and type 2 diabetes, whilst reduction in food intake, by increasing insulin sensitivity and improving glucose homeostasis, is currently recommended in the treatment of these metabolic disorders [14 ]. such a caloric restriction may include a relative decrease of food intake [57 ] or otherwise either a total short [8, 9 ] or prolonged fasting. intermittent overnight fasting, inspired by the daily fasting period during the ramadan, was recently reported to prevent the progressive deterioration of glucose tolerance otherwise taking place in sand rats exposed to a hypercaloric diet [1113 ]. the major aim of the present study was to investigate whether a comparable benefit of intermittent fasting may prevail in streptozotocin - induced diabetic rats. eight to 10 weeks after birth, female wistar rats (charles river, wilmington, ma, usa) were injected intraperitoneally, after overnight starvation, with streptozotocin (stz, 65 mg / kg body wt.) freshly dissolved in a citrate buffer (50 mm, ph 4.5). these rats were given access during the night after the injection of streptozotocin to a solution of saccharose (10 g/100 ml) to prevent possible hypoglycemia. five days after the injection of streptozotocin, the glycemia was measured with the help of glucometer (lifescan benelux, beerse, belgium) in blood obtained from caudal vein. only those rats displaying a glycemia in excess of 16.7 mm were kept for further investigations. in order to compare the effects of an intermittent fasting, mimicking the ramadan fasting, to that of a caloric restriction, twenty days after the injection of either streptozotocin (stz rats) or the citrate buffer vehicle (control rats), the rats were either given free access to food throughout the experimental period (nf : nonfasting rats), deprived of food and water from 5 p.m. to 8 a.m. (if : intermittently fasting rats) or given access from 5 p.m. onwards to an amount of food comparable to that ingested by the if rats (cr : calorie - restricted rats). relative to the food intake in nf rats, such a caloric restriction represented a 20% decrease in food intake in the control animals and a 40% decrease of food intake in the stz rats. the initial body weight was measured before the injection of streptozotocin or its citrate buffer vehicle, 7 days thereafter, 20 days thereafter, on day 4, 7, 11, 14, 18, 21, and 27 of the final 30 days experimental period and at sacrifice, after overnight starvation. likewise, food intake was measured 15 to 20 days (6 measurements) after injection of stz or its vehicle in 6 control rats and 3 groups of 5 - 6 stz rats, and daily (26 measurements) during the last 30 days experimental period. an ipgtt [14, 15 ] was conducted in all rats on day 10, 20, and 29 of the final 30 days experimental period, after overnight starvation. a solution of d - glucose (20%, w / v) in distilled h2o was intraperitoneally injected in conscious rats in order to deliver 2 g d - glucose per kg body weight. the glycemia was measured by a glucometer before and 30, 60, and 120 min after the administration of d - glucose in blood samples obtained from a caudal vein. the total and incremental areas under the glycemic curve (auc) were computed in each individual experiment. at the end of the experimental period, the rats were sacrificed after overnight starvation and under anesthesia provoked by the intraperitoneal injection of a solution containing ketamine and xylocaine. blood samples were obtained from the heart and placed in heparinized tubes, the plasma being then separated by centrifugation and stored at 80c. the plasma d - glucose and insulin concentrations were measured by methods described in the cited references. these measurements were used to calculate the insulinogenic index (i.e., the ratio between the plasma insulin concentration, expressed as mu / l, and the difference between the plasma d - glucose concentration, expressed as mm, and 4.0 mm, considered as the threshold value for stimulation of insulin secretion by the hexose) and the homa index (i.e., the product of the plasma insulin concentration, expressed as u / ml, times the plasma d - glucose concentration, expressed as mm). the pancreas were either used for the isolation of islets or fixed for immunohistochemical examination. groups of 4 islets each, obtained by the collagenase procedure, were incubated at 37c for 90 min in 0.5 ml of a salt - balanced medium containing bovine serum albumin (5 mg / ml) and equilibrated against a mixture of o2/co2 (95/5, v / v). the insulin released by the islets during incubation and their final insulin content were measured by radioimmunoassay. for immunodetection of insulin, pancreatic rehydrated paraffin sections were blocked 1 h at room temperature with 1 : 20 normal goat serum (vector laboratories, burlingame, ca, usa) in pbs for nonspecific reactions. the slides were incubated with primary anti - insulin (12018, sigma - aldrich, st louis, mo, usa) mouse monoclonal antibody overnight at 4c at a concentration of 1/3000 in normal goat serum (1/20 in pbs). the secondary antibody, rhodamine red x - conjugated goat anti - mouse igg (h + l) (115 - 295 - 146, jackson immunoresearch laboratories, west grove, pa, usa) was applied at a dilution of 1/200 in pbs / normal goat serum for 30 min at room temperature. the slides were mounted, and dna was counterstained with dapi (in vitrogen, merelbeke, belgium). the staining patterns were observed with an axioplan and recorded with an axiocam (carl zeiss, oberkochen, germany). the slides were incubated overnight at 4c with the first antibody : anti - insulin (santa cruz biotechnology, inc., ca, usa) at dilution 1 : 500 in pbs with appropriate blocking serum at a dilution of 1/20. purified immunoglobulins (igg) (sigma - aldrich, st louis, mo, usa) from nonimmunized rabbit were used as negative controls. the slides were further incubated with the secondary biotinylated antibody : goat anti - rabbit igg (h+l) (ba-1000, vector laboratories) at a dilution of 1/300 in pbs for 30 min, at room temperature. -cell mass was measured by point - counting morphometry on these immunoperoxidase - stained sections. the measurement was performed on live using leica microsystems microscope (heerbrugg, switzerland). a grill of 110 points was used to assess insulin positive stained islet on each field. individual -cell area was determined by using image j logician on immunofluorescence stained sections of pancreas used for -cell apoptosis assessment. the -cell area was calculated from the ratio between individual area and -cell nuclei number within the area taken in consideration. for glucagon immunodetection, the same procedure as that described for insulin immunodetection by the abc - dab technique was used. the sole difference consisted in the first antibody, that is, antiglucagon (a0565, dako, carpinteria, ca, usa) used at dilution 1 : 400. the quantification of -cell apoptosis by the tunel method was performed using the in situ cell death detection kit, pod (roche diagnostics, vilvorde, belgium). at the end of this procedure, the pancreatic sections were rinsed with pbs and eventually exposed overnight at 4c to primary anti - insulin antibody (see above) followed by exposition for 30 min at 20c to the rhodamine red x - conjugated secondary antibody (1/200 dilution). the apoptotic index represents the ratio between positive and total nuclei of insulin - producing cells in each islet. all results are presented as mean values (sem), together with the number of separate determinations (n). the statistical significance of differences between mean values was assessed by use of student 's t - test. at day zero of the last 30 days experimental period, the mean body weights of the control and stz rats did not differ significantly (p > 0.52) from one another, with an overall mean value of 223 4 g (n = 33). over the 2 weeks following the injection of either streptozotocin or the citrate buffer vehicle, the changes in body weight averaged + 17.6 4.0 g (n = 16) in control rats, as distinct (p 0.4 or more) from those recorded in the same type of rats (control or stz) over the 2-week period following the injection of streptozotocin or the citrate buffer vehicle. over the last 30 days of the experiments, the gain in body weight was much lower (p 0.77) in control rats (6.31 0.62 mm ; n = 48) and stz rats (6.68 1.15 mm ; n = 52). the profile of glycemia during the ipgtt conducted in control rats is illustrated in figure 2. the total auc averaged in the if and cr control rats, respectively, 94.0 3.9% (n = 16) and 96.1 2.8% (n = 17) of the mean corresponding values recorded on the same day in the fed control rats (100.0 5.0% ; n = 15). none of these mean values differed significantly from one another (p > 0.35 or more). however, as documented by the data listed in table 2, the incremental auc tended to be lower in if and cr control rats than in fed control rats. thus, the values recorded in if and cr control rats, respectively, averaged 59.1 15.3% (n = 16) and 65.3 9.5% (n = 17) of the mean corresponding values recorded on the same day in the fed control rats (100.0 15.1% ; n = 15). such a difference only achieved statistical significance (p 0.24) being observed between the latter two groups of stz rats (table 3). likewise, the total auc was lower (p 0.21) from one another. the incremental area, however, was not significantly different (p > 0.08 or more) in if rats, as compared to either nf or cr rats, being only significantly higher (p 0.49 or more) in nf, if, and cr animals (table 4). in the stz rats, however, the overall mean value found in the if and cr animals (24.63 2.91 ; n = 12) was significantly lower (p 0.26 or more) from that found in the nf animals. such was also the case (p > 0.15 or more) in the stz rats. as illustrated in figure 4, no significant correlation was observed between plasma insulin and d - glucose concentration in the 15 control animals (r = + 0.0416 ; p > 0.1), whilst a highly significant negative correlation between these two variables prevailed in the 17 stz rats (r = 0.6892 ; p 0.25) or elevation (f = 2.156 ; f = 1,28 ; p > 0.1). the insulinogenic index was much higher (p 0.23) from those found in the nf control rats (table 5). in the stz rats, however, both the relative and absolute values for -cell mass were higher (p 0.66) in control animals (0.84 0.09 g ; n = 9) and stz rats (0.79 0.04 g ; n = 9). in both cases, however, and relative to the mean values found in nf animals (100.0 2.8% ; n = 6), those recorded in the if rats (68.9 7.5% ; n = 6) were significantly lower (p 0.24) and 123.9 4.3% (n = 40 ; p 0.59) from one another. figure 8 illustrates the immunodetection of -cells using rhodamine - labelled secondary antibody, and figure 9 the immunodetection of apoptotic -cells by the tunel procedure. in the control animals, the percentage of apoptotic islet -cells was comparable in nf, if, and cr rats, with an overall mean value not exceeding 4.31 0.10% (n = 15), as distinct (p 0.49) was observed in terms of the changes in body weight of the stz rats over the 30 days final experimental period, when comparing nf animals (12.4 2.8 g ; n = 5) to if rats (18.8 7.9 g ; n = 6). no statistically significant beneficial effects of caloric restriction in the stz rats was observed when comparing nf to cr diabetic animals. moreover, the decrease in body weight observed in the stz rats during the final 30 days experimental period was 2.5 to 3.8 times higher (p < 0.001) in cr rats than in if and nf animals, respectively. even in control rats, the gain in body weight was much lower in cr animals than in if ones (figure 1). this coincided with lower mean values for the plasma insulin concentration, insulinogenic index, and homa index in if control rats than in cr control rats examined at sacrifice after overnight starvation (table 4). thus, for these three variables, the values recorded in if control rats averaged 71.6 8.6% (n = 15 ; p < 0.06) of the mean corresponding values found in cr control rats (100.0 11.5% ; n = 15). since such distinctions between if and cr control rats could not be ascribed to any difference in either food intake or the responsiveness to d - glucose of isolated pancreatic islets incubated in vitro, they suggest a more stressful situation in cr control rats than in if control animals. to a large extent, a comparable situation may prevail in cr as distinct from if diabetic animals. in conclusion, therefore, the present study allows to extend to streptozotocin - induced diabetic rats, the proposal that intermittent fasting exerts a beneficial effect on glucose tolerance [1113 ]. in our opinion, such a dietary approach merits to be also considered as a possible approach to prevent or minimize, if not correct, disturbances of glucose homeostasis in human subjects.	this study investigates the effects of intermittent overnight fasting in streptozotocin - induced diabetic rats (stz rats). over 30 days, groups of 5 - 6 control or stz rats were allowed free food access, starved overnight, or exposed to a restricted food supply comparable to that ingested by the intermittently fasting animals. intermittent fasting improved glucose tolerance, increased plasma insulin, and lowered homeostatis model assessment index. caloric restriction failed to cause such beneficial effects. the -cell mass, as well as individual -cell and islet area, was higher in intermittently fasting than in nonfasting stz rats, whilst the percentage of apoptotic -cells appeared lower in the former than latter stz rats. in the calorie - restricted stz rats, comparable findings were restricted to individual islet area and percentage of apoptotic cells. hence, it is proposed that intermittent fasting could represent a possible approach to prevent or minimize disturbances of glucose homeostasis in human subjects.
myiasis is a term derived from the greek word myia, meaning invasion of vital tissue of humans or other mammals by fly larvae.[15 ] the term myiasis hope in 1940, and since then, it has been used to designate infestation by the larvae.[68 ] myiasis was defined by zumpt as the infestation of live human and vertebrate animals by dipterous larva, which at least for a certain period feed on host 's dead or living tissue, liquid body substances or ingested food. a condition similar to myiasis was considered by the hindu mythology as god 's punishment to sinners. a 22-year - old male patient reported to the outpatient department of oral medicine with a primary complaint of worms in the mouth since 4 days. the medical history revealed that the patient was affected by cerebral palsy since birth [figure 1 ]. clinical oral examination revealed an area of ulceration in the anterior palatal aspect of the mouth, accompanied by erythema, redness and bleeding on probing [figure 2 ]. external profile of the patient intraoral photograph showing palatal swelling a total of 20 larvae were grasped gently and taken out with the help of tweezers after application of turpentine oil. out of these, only three were viable. these were then taken to department of parasitology where they were identified as larvae of the common housefly. histopathological examination of the incisional biopsy specimen revealed dense inflammatory infiltrate comprising chiefly lymphocytes in the connective tissue stroma. this parasitic infestation frequently occurs in rural areas, infecting livestock and pets such as dogs and cats. after the fly lays eggs in the dead and decaying tissues, the larvae hatch in about 810 hours, soon after which they burrow into the surrounding tissues ; and in this stage, there will be tissue inflammation ensuing discomfort, which makes the patient consult a doctor. the opening of the burrow is usually kept patent with induration of the marginal tissues, and is raised forming a dome - shaped warble. they position their heads down so that the posterior spiracles could become exposed to the open air to make respiration possible. after the young larvae penetrate the skin of the host, they take 812 days to develop into the prepupal stage and then leave the host to pupate. while in human skin the most common anatomic sites for myiasis are the nose, eye, lung, ear, anus, vagina and, more rarely, the mouth. incidence of oral myiasis as compared to that of cutaneous myiasis is less as the oral tissues are not permanently exposed to the external environment. it has been reported among epilepsy patients with lacerated lips following a seizure, incompetent lips and thumb sucking habits, advanced periodontal disease, at tooth extraction sites and patients with tetanus with mouth propped open to maintain their airway. the stage of larvae lasts for 68 days during which they are parasitic to human beings. the larvae have backward directed segmental hooks with which they anchor themselves to the surrounding tissue. they are photophobic and tend to hide deep into the tissues for a suitable niche to develop into pupa. this unusual type of gingival myiasis may occur in an unconscious or sleeping person when the mouth is left open. periodontal disease of the oral cavity, with pockets, provides a perfect environment for the eggs to hatch and for the larvae to grow in the warm and moist conditions. histological examination of tissue has been reported to show evidence of dense inflammation, dystrophic calcification, and foreign bodies compatible with features of abscess. the standard treatment of myiasis is manual removal with hemostatic or clinical pincers, associated with or without the administration of topical asphyxiation drugs, which forces the larvae to come out. it is important to remove all the larvae, otherwise the cavity does not heal properly and can also become chronically infected. various substances (ether, chloroform, olive oil, calomel, iodoform, phenol mixture) have been recommended ; however, they were found to produce controversial results. ivermectin, a semi - synthetic macrolide antibiotic, is found safe for human use as proposed by shinohara. and osorio. in some cases to conclude, myiasis affects mostly the uncovered body areas where oviposition is easily carried out. it frequently affects low socioeconomic level individuals with poor hygiene habits and unhealthy patients with psychiatric disorders, diabetics, and immunocompromised patients. undoubtedly, preventive approach measures, including basic health care, hygiene, access to primary health service, and safe water and drainage, are fundamental to prevent cases such as this one.	myiasis is a relatively rare condition arising from the invasion of body tissues or cavities of living animals or humans by maggots or larvae of certain species of flies. it is an uncommon clinical condition, being more frequent in underdeveloped countries and hot climate regions, and is associated with poor hygiene, suppurative oral lesions ; alcoholism and senility. its diagnosis is made basically by the presence of larvae. the present article reports a case of oral myiasis involving 20 larvae in a patient with neurological deficiency.
less than one percent of women living with hiv have perinatally acquired hiv infection (phiv). according to recent centers for disease control and prevention (cdc) hiv surveillance data, approximately 2,388 phiv women are living in the united states. due to lifelong hiv infection, many women with phiv have been exposed to multiple antiretroviral (arv) therapy regimens. these exposures may include inadequate therapy during periods when there were limited arv therapy options, such as mono and dual therapy. as a consequence of suboptimal therapy, inconsistent drug adherence, and/or prolonged intermittent exposure to multiple arv classes, phiv women may have hiv that has developed significant drug resistance [25 ]. antiretroviral drug resistance can limit options for therapy during pregnancy and potentially complicate obstetrical care for hiv - infected women. phiv women who have developed arv drug resistance may require arv therapies which are less well studied in pregnancy and may have unknown toxicities [6, 7 ]. potentially secondary to noncompliance or a suboptimal arv regimen, phiv patients may have poor viral suppression during pregnancy resulting in cesarean delivery and a higher risk of perinatal transmission [3, 4, 8, 9 ]. examples of other risks specific to phiv pregnant women include complex psychosocial issues, unplanned pregnancies, and transmission of hiv to susceptible partners [3, 6, 10, 11 ]. arv resistance rates have been reported as high as 3050% in phiv pregnant women [4, 9, 12 ]. arv resistance mutations and drug classes affected are not well described in previous studies of phiv pregnant women [4, 9, 12 ]. the primary objective of this study was to determine if phiv pregnant women are more likely than pregnant women with nonperinatal hiv infection (nphiv) to have arv drug resistance. we describe the arv classes affected by hiv genotypic mutations in both phiv and nphiv women. our secondary objective is to describe and compare potential adverse maternal and neonatal outcomes among phiv and nphiv pregnant women. the primary objective of this study was to determine how much more likely phiv women are to have hiv genotypic mutations that confer clinically significant arv resistance during pregnancy compared to nphiv women. prior data indicated the probability of genotypic resistance to arvs, specifically nonnucleoside reverse transcriptase inhibitors (nnrtis), in drug nave nphiv pregnant women to be 1317%. based on previous reports, phiv women may have arv resistance rates as high as 50%. we anticipated a noncollinear relationship between arv resistance and timing of hiv infection. in order to demonstrate odds ratio of at least 4.0 (13% 4 = 52%) for arv resistance in phiv pregnant women relative to nphiv women, 41 cases (phiv) and 41 controls (nphiv) were required to reject the null hypothesis that this odds ratio equals one with a probability of 0.8. the type i error probability associated with this test of the null hypothesis was 0.05. the study was not powered to determine differences in pregnancy outcomes between phiv and nphiv. because phiv is a rare condition in pregnancy, a multisite, retrospective cohort study was conducted to enroll the necessary sample size of phiv participants. to identify potential study sites, an email was sent to all providers participating in the reproductive infectious diseases listserv (reproidhiv listserv). collaborators from twenty - two sites responded with interest and were provided a copy of the protocol. seven sites at academic medical centers in british columbia, california, colorado, missouri, new york, pennsylvania, and south carolina elected to participate and the study protocol was approved by the institutional review boards at each site (irb # 13184). pregnant women with phiv who received prenatal care at any of the study sites from 2000 to 2014 were eligible for participation. a woman was considered to have phiv if her hiv serostatus was confirmed and determined to be acquired from her biological, serostatus - confirmed hiv - infected mother in the absence of any other risk factors (i.e., blood transfusion). control participants were identified as pregnant women with nphiv receiving prenatal care during the study period at any study site. a woman was considered to have nphiv if she was diagnosed with hiv at 11 years of age in the absence of questionable perinatal infection (hiv - infected mother) or other risk factors for childhood infection (breastfeeding from an hiv - infected mother or blood transfusion). nphiv participants were selected based on a similar age to study participants (1 year of age). participants were age - matched in order to reduce an uneven distribution of age - related medical comorbidities, such as preexisting hypertension and diabetes, which may potentially impact pregnancy outcomes. the medical records of participants were reviewed by site specific investigators to obtain data from maternal antepartum, intrapartum, and postpartum care. maternal variables included age, race, ethnicity, marital status, insurer, current partner 's hiv status, history of opportunistic infections, existing medical and psychiatric diagnoses, gestational age at entry into prenatal care and at delivery, hiv rna viral load (copies / ml) and cd4 cell count (cells / mm) at entry into prenatal care and at delivery, arv regimens before, during, and after pregnancy, hiv genotypic mutations associated with clinical drug resistance, evidence of sexually transmitted infections ((stis) : neisseria gonorrhea, chlamydia trachomatis, trichomonas vaginalis, treponema pallidum, human papilloma virus, hepatitis b and c, and herpes simplex virus), number of prenatal visits, antepartum complications, intrapartum prophylaxis iv zidovudine (azt) when indicated, mode of delivery, postpartum infections including chorioamnionitis, and birth outcomes (e.g., live birth, intrauterine fetal demise, and spontaneous or elective abortion). maternal antepartum complications of interest were hypertensive disorders of pregnancy, diabetes, maternal infection(s), preterm labor, anemia, and fetal anomaly and/or aneuploidy. in participants who gave birth to a live infant, data were collected from neonatal records up to 18 months of age. variables of interest included birth weight, apgar scores, level of nursery admit, neonatal postexposure arv prophylaxis, duration of arv prophylaxis, and hiv status. neonates were considered hiv - infected if at least two positive hiv dna pcr tests were confirmed before 18 months of age. to protect confidentiality, study site investigators collected and managed data using redcap (research electronic data capture), a secure, web - based application designed to support data capture. continuous variables were compared using student 's t - test (means) and wilcoxon rank - sum tests (medians). continuous variables were tested for normality, and medians were compared when data were not normally distributed. univariate logistic regression was used to determine factors associated with the presence of arv drug resistance. as determined by the sample size calculation, 41 phiv and 41 nphiv women were included in the analysis (figure 1). the mean age of participants at the time of pregnancy was 21 years (standard deviation (sd) the median parity of women was one (interquartile range (iqr), 0 - 1). the median gestational age at which women presented for prenatal care was 11 weeks (iqr, 714 weeks), and the mean number of prenatal visits prior to delivery was 10 (sd 5). the hiv status of the participants ' male partner was recorded in approximately half of the women. nphiv women were more likely to report an hiv - infected sexual partner(s) than phiv women (32% versus 4%, p = 0.02). when comparing phiv and nphiv women, there were no differences in race, ethnicity, age, and prenatal care initiation or duration. nphiv women were more likely to be parous than phiv women (1 (iqr 04) versus 0 (iqr 02), p = 0.0004), and phiv women were more likely to have a history of abnormal cervical cytology (50% versus 27%, p = 0.03). the mean duration of known hiv infection for phiv women was 21 (sd 4) years and it was 2 (sd 3) years for nphiv women (p < 0.0001). of the participants with nphiv infection, 21 were diagnosed with hiv within one year of pregnancy and 20 were known to have hiv of duration of more than one year. forty - three percent of all participants (36/82) reported current arv use at initial presentation for prenatal care. phiv women were more likely to report taking arvs at presentation (68% versus 23%, p = 0.006). although only 27 phiv women were on arvs at conception, all phiv women in this study were exposed to arvs during their lifetime prior to enrollment. the specific arv regimens prescribed to individual subjects throughout their lives prior to enrollment in this study are not available for inclusion in this study. of the nphiv participants with hiv diagnosis greater than one year the median hiv rna viral load (copies / ml) and mean cd4 cell count (cells / mm) collected within three months of the initial prenatal visit were not significantly different between phiv and nphiv women. of the women reporting arv use at their initial pregnancy visit, phiv women were more likely than nphiv women to have hiv rna viral load 1,000 copies / ml (46% versus 0, p = 0.01) (tables 1 and 2). over half of participants (24 phiv and 25 nphiv) had an hiv rna viral load 1,000 copies / ml at their initial prenatal evaluation. when using an hiv rna viral load 1,000 copies / ml as criteria for collecting a genotype (hiv-1 genotype, viroseq, arup laboratories, salt lake city, ut), 60% of participants would have been eligible for genotypic testing for hiv drug resistance. not all participants who were eligible for resistance testing had a genotype collected within three months of their initial pregnancy visit. collection of an hiv genotype during this time period was reported in 34 (42%) participants. although similar numbers of phiv and nphiv women met criteria for resistance evaluation by genotype (24 phiv and 25 nphiv), phiv participants were more likely to have genotypic testing collected within three months of their initial pregnancy visit compared to nphiv counterparts (22 phiv (54%) and 12 nphiv (29%), p = 0.03). when accounting for genotype collection within three months of presentation for prenatal care, 55% phiv versus 17% nphiv had drug resistance (p = 0.03) (figure 2). in addition to genotype resistance noted in 12 phiv and two nphiv women within three months of initial prenatal care, arv drug resistance was documented for seven additional participants either before pregnancy or during pregnancy. arv drug resistance was documented in 21 participants (17 phiv and four nphiv), but 18 resistance patterns were available for analysis (15 phiv and 3 nphiv participants). multiclass arv drug resistance, resistance to more than one arv class, occurred exclusively in phiv women (16% versus 0, p = 0.03). genotypic resistance to multiple arv drug classes was documented in 6 phiv women (nrtis n = 6, nnrtis n = 11, and protease inhibitors (pis) n = 6). nphiv women had resistance to nrti (n = 1) and nnrtis (n = 2), but no pis resistance was noted. arv regimens were adjusted during pregnancy in seven women (five phiv and two nphiv) secondary to drug resistance (table 3). univariate analysis was performed to identify which maternal factors were associated with the arv resistance mutations. among women who had an hiv genotype collected, phiv infection was associated with an increased risk of drug resistance (or 6.0 (95% ci, 1.0334.8), p = 0.05). phiv infection was the only variable with a statistically significant association to arv drug resistance. other variables analyzed were the duration of hiv infection prior to pregnancy, elevated hiv viral load at presentation for pregnancy, medical comorbidities, race / ethnicity, history of psychiatric illness, and maternal age. the majority of participants had documented arv use during pregnancy (100% phiv and 93% nphiv, p = 0.24) (table 2). phiv women were more likely to take integrase inhibitors (20% versus 2%, p = 0.03). fusion inhibitors (n = 3) and ccr5 antagonists (n = 1) were used exclusively in phiv women. resistance testing and results for these alternative drug classes were not available for analysis. at the time of delivery, there was no significant difference in the number of women from either group taking nrtis, nnrtis, and pis (table 3). an appendix in supplementary material available online at http://dx.doi.org/10.1155/2016/4897501 which provides a detailed description of the types of arvs prescribed before, during, and after pregnancy for all study participants is available upon request. psychiatric illness was more common in women with phiv (50% phiv versus 27% nphiv, p = 0.03). depression was the most common psychiatric diagnosis, affecting 43% of phiv and 22% of nphiv women. nphiv and phiv women had similarly high rates of medical comorbidities in pregnancy (46% nphiv versus 29% phiv, p = 0.1). the most common medical comorbidities reported were asthma, obesity, chronic hypertension, and anemia. rates of stis were similar between phiv and nphiv women (p = 0.1). the following stis were common among participants in both groups : genital herpes (68%), t. vaginalis (22%), c. trachomatis (14%), and n. gonorrhoeae (3%). chronic hepatitis was infrequent among participants (6%) ; three women had hepatitis b and two had hepatitis c. both groups had similar rates of pregnancy - related complications (45% versus 59%, p = 0.2). the most common complications of pregnancy were hypertensive disorders of pregnancy (12%), preterm labor / shortened cervical length / preterm delivery (20%), and premature rupture of membranes (2%) (tables 1 and 2). live birth rates and cesarean delivery rates were similar among phiv and nphiv women ((97% phiv versus 98% nphiv, p = 1.0) and (47% phiv versus 46% nphiv, p = 0.9), resp.). at the time of delivery, the proportions of participants with hiv rna viral load 1,000 copies / ml and hiv rna viral loads below the level of detection (< 40 copies / ml) were similar between groups ((26% phiv versus 28% nphiv, p = 0.9) and (56% phiv versus 63% nphiv, p = 0.5), resp.). the difference in the proportion of participants with hiv rna viral loads between 40 and 999 copies / ml near delivery was not statistically significant (18% phiv and 9% nphiv, p = 0.18). similar proportions of participants received at least three hours of intrapartum iv azt when indicated (80% versus 84%, = 0.7) (table 2). the median gestational age at the time of delivery was 38 weeks in both groups. preterm birth (< 37 weeks gestation) rates were not significantly different between groups (11% phiv versus 28% nphiv, p = 0.08). median birth weights were lower in the nphiv women (2,742 (iqr 2,4353,200) versus 3,065 (iqr 2,6593,370), p = 0.02), but the proportion of low birth weight (< 2,500 gm) infants was similar (34% nphiv versus 22% phiv, p = 0.2). neonatal intensive care unit admissions were not significantly different between groups (29% nphiv versus 14% phiv, p = 0.2). hiv perinatal transmission was documented among two infants (2/32, 6%) born to a single nphiv mother one year apart. the mother did not have prenatal care and did not take arvs during either pregnancy. both infants were born preterm, one by spontaneous vaginal delivery complicated by previable premature rupture of membranes and chorioamnionitis and the other by emergent cesarean delivery in the setting of abruption and preeclampsia. there were no documented cases of hiv perinatal transmission among women with phiv (table 2). hiv genotypic patterns suggestive of clinically relevant arv drug resistance were documented in phiv pregnant women three times more frequently than nphiv pregnant women. previous studies have documented clinically relevant genotypic mutations in 3050% of phiv pregnant women and in 1317% of nphiv pregnant women [4, 9, 12 ]. we anticipated and found comparatively higher rates of arv drug resistance among phiv women likely due to their lifelong hiv infection and potential intermittent exposure to multiple arv classes and suboptimal arv regimens. providers caring for hiv - infected pregnant women should be aware of the potential for high rates of arv resistance among all pregnant women in this age group but especially among women born with hiv infection. compared to previous studies, we were able to document the patterns of hiv drug resistance in phiv pregnant women. the genotypic resistance in nphiv women was limited to the nrti and nnrti classes compared to multiple arv classes in phiv women. six phiv women had multidrug resistant hiv infection, and one phiv women had resistance mutations to all three major arv classes (nrti, nnrtis, and pis). due to multidrug resistance, phiv women were more likely to be prescribed arv combinations that are nonstandard regimens for the prevention of perinatal transmission. during the study period, nonstandard arv therapies prescribed for phiv given the importance of hiv viral suppression during pregnancy, providers caring for pregnant women with phiv should be familiar with the potential use and limitations of these alternative arvs during pregnancy. our findings of high rates of arv drug resistance support the recommendation for hiv genotype analysis in early pregnancy. genotypic testing should be collected and assessed as early as possible in all hiv - infected women. these tests should be repeated during later pregnancy in women with poorly suppressed hiv rna viral loads. when resistance to standard arv classes used for prevention of perinatal transmission is suspected, providers should consider alternative drug classes and order additional resistance testing for integrase inhibitors, fusion inhibitors, or ccr5 antagonists. phiv women were more likely to report arv use at the time of pregnancy diagnosis, but overall use of arvs prior to conception was low. only 68% of phiv women and even fewer nphiv women (23%) were on arv therapy at initial presentation for pregnancy care. only 30% of nphiv women diagnosed with hiv at least one year prior to pregnancy reported arv use at presentation for care. in previous studies, phiv women were more likely to have poor viral suppression during pregnancy, likely due to inconsistent drug adherence [35, 8 ]. in contrast to these reports, phiv women in our study had similar rates of virological suppression and comparable cd4 cell counts to nphiv women during pregnancy. providers caring for reproductive age women with hiv infection should be aware of their pregnancy intentions in order to reduce the risk of perinatal hiv transmission from delayed exposure to effective arv therapy before and during pregnancy. although the reduction of perinatal transmission is a primary objective of prenatal care for hiv - infected women, pregnancy is also a time when hiv - infected women are examined frequently and can be screened for other significant medical comorbidities. current or past psychiatric illness, especially depression, was more common among phiv women (50%). given the potential effects of depression on pregnancy, hiv - infected women should receive a multidisciplinary approach including psychiatric evaluation and support services [10, 18 ]. additionally, phiv women were more likely to have a history of abnormal pap smears. frequent visits during pregnancy can provide adequate time for evaluation for cervical cancer risks among hiv - infected women. our study revealed that hiv - serodiscordance was very common among pregnant women and their partners. phiv women were more likely to have hiv susceptible partners (96%) compared to nphiv women (68%). this difference is likely explained by nphiv women potentially having acquired hiv from their current partners, as compared to phiv counterparts, who acquired hiv at birth. prenatal providers should be aware of the potential risk of hiv transmission to hiv susceptible partners during pregnancy. in an effort to reduce the transmission of hiv to susceptible partners, providers should provide risk reduction counseling to all serodiscordant couples and frequent hiv testing of susceptible partners and evaluate partners for hiv preexposure prophylaxis. this study design was necessary given the relative rarity of the exposure of phiv in pregnancy. this can partially be explained by 33 (40%) women having hiv rna viral loads < 1,000 copies / ml at the time of initial pregnancy care. it is unclear to the investigators why genotypes were not collected in all 49 women who were eligible for hiv drug resistance evaluation (hiv rna viral load 1,000 copies / ml). another limitation of our study is that we neglected planning for and carrying out collection of information regarding substance use, including tobacco, alcohol, and prescription and/or street drugs. a prospective study design and inclusion of all phiv and nphiv women presenting to any site during the study period would be the most effective means for evaluating the primary outcome and potential differences in pregnancy outcomes. however, given the rarity of phiv during pregnancy, a prospective, multisite study would exceed the resources available. the majority of study participants were identified at an academic center in the southeast, where the principal investigator practices. according to the cdc, the southeast has the 2nd highest prevalence of hiv infection in the united states. as of 2011, the southeast had the highest number of new hiv infections and the largest proportion of individuals living with stage 3 hiv / aids. perinatal transmission rates are also high in this region. due to the increased rate of perinatal hiv transmissions, it is reasonable that the majority of cases and controls are clustered in this region due to prevalence of infection. however, the entirety of phiv cases were distributed from multiple locations in north america suggesting this data has generalizability for many centers caring for phiv women during pregnancy. this is the largest single group of phiv women studied in comparison to age - matched nphiv controls. this study adds to the medical literature by describing the types of hiv arv mutations that affect the care of hiv - infected women during pregnancy. based upon the data presented here, obstetric providers of phiv and nphiv women should have a high suspicion for clinically relevant hiv drug resistance early in pregnancy in order to best select effective therapies for the prevention of perinatal hiv transmission. lastly, phiv women did not experience higher rates of perinatal complications and there were no perinatal hiv transmissions to phiv - exposed infants. although not powered to identify any significant differences in pregnancy outcomes, our findings suggest that phiv women may have similar pregnancy outcomes compared to nphiv. further investigation is needed, but providers can offer some reassurance to women living with phiv infection that they are likely to have pregnancy outcomes comparable to other hiv - infected pregnant women.	objective. to compare hiv drug resistance in pregnant women with perinatal hiv (phiv) and those with nonperinatal hiv (nphiv) infection. methods. we conducted a multisite cohort study of phiv and nphiv women from 2000 to 2014. sample size was calculated to identify a fourfold increase in antiretroviral (arv) drug resistance in phiv women. continuous variables were compared using student 's t - test and wilcoxon rank - sum tests. categorical variables were compared using 2 and fisher 's exact tests. univariate analysis was used to determine factors associated with antiretroviral drug resistance. results. forty - one phiv and 41 nphiv participants were included. women with phiv were more likely to have drug resistance than those with nphiv ((55% versus 17%, p = 0.03), or 6.0 (95% ci 1.034.8), p = 0.05), including multiclass resistance (15% versus 0, p = 0.03), and they were more likely to receive nonstandard arvs during pregnancy (27% versus 5%, p = 0.01). phiv and nphiv women had similar rates of preterm birth (11% versus 28%, p = 0.08) and cesarean delivery (47% versus 46%, p = 0.9). two infants born to a single nphiv woman acquired hiv infection. conclusions. phiv women have a high frequency of hiv drug resistance mutations, leading to nonstandard arvs use during pregnancy. despite nonstandard arv use during pregnancy, phiv women did not experience increased rates of adverse pregnancy outcomes.
odontogenic cysts are classifed by the world health organization as infammatory and developmental according to their epithelial lining. lateral periodontal cysts (lpc) have been regarded as an independent condition [1 - 4 ]. lpcs are defined as nonkeratinized and noninflammatory developmental cysts located adjacent or lateral to the root of a vital tooth. this cyst 's most frequent location is at the level of mandibular premolars but it has been reported occurring in the other areas. lpc is one of the cysts of lower incidence among developmental odontogenic cysts. since pain or other clinical symptoms have seldom been reported, the lesion is often discovered on routine radiographic examination. radiographs of the lateral periodontal cyst show a well - circumscribed round or ovoid radiolucent area, usually with a sclerotic margin. most of them are less than 1 cm in diameter [5 - 8 ]. histologically, the lateral periodontal cyst is a distinct type of developmental cyst characterized by a thin, nonkeratinized epithelium usually 1 to 5 cell layers thick, which resembles the reduced enamel epithelium. the epithelial lining exhibits focal thickenings or plaques, in which clear glycogen - containing epithelial cells have often been found. it presents unique characteristics and there is an important differential diagnosis with lesions of endodontic and periodontal origin, and other cysts from the same group [5 - 8 ]. the pathogenesis of lpc, the gingival cyst of the adult, the botryoid odontogenic cyst and the glandular odontogenic cyst may be correlated. some authors, based on the clinical and morphological similarities, reinforce that the gingival cyst of the adult and lpc present a common histogenesis, and these lesions may have the same extra - osseous and intra - osseous characteristics, respectively. this paper reports a classic case of lpc which is located in the anterior region of mandible, and also presents a brief literature review of the clinical, radiological and histopathological features of lpc. a 50 year old black female patient was referred to so paulo state university - unesp, so jos dos campos dental school, department of biosciences and oral diagnosis, so jos dos campos, so paulo, brazil, complaining of mandibular left lateral incisor mobility. the patient reported that she had used the stick to tooth in this region, shortly before the change was observed, approximately six months ago. the palpation of vestibular surface of the alveolar process in the region of teeth # 32 and # 33 was asymptomatic. radiographic examination revealed a well - circumscribed, unilocular, radiolucent area with approximately 0.5 cm in diameter adjacent to the roots of the left mandibular lateral incisor and canine (figure 1). periapical radiograph showing circumscribed radiolucent area located between two vital teeth # 32 and # 33. on the basis of these findings, a total enucleation of the lesion was realized using a surgical curette (figure 2). histological sections showed a single cavity lesion lined by epithelium of variable thickness, sometimes displaying one or two layers of cuboid cells and sometimes showing areas that formed thick clusters of cells more voluminous in the midst of which several pas positive clear cells were observed. the capsule consisted of fibrous tissue with several bleeding areas and absence of inflammation (figure 3 b, c and d). the histological findings supported the diagnosis of lateral periodontal cyst of developmental origin. a = photograph showing macroscopic aspects of the lesion. b = histopathologic view of the lesion shows one cystic cavity only ; the capsule of fibrous tissue showed several bleeding areas and absence of inflammation (hematoxylin and eosin stain, original magnification x25). c = histopathologic view of the lesion shows epithelium lining rich in cells with glycogen (hematoxylin and eosin stain, original magnification x100). d = histopathologic view of the lesion shows pas positive clear cells (pas x100). follow - up of the case, twenty four months after operation, showed uneventful healing and spontaneous regeneration of bone in periapical radiography. lpc is considered as developmental odontogenic cyst with unusual occurrence that may be associated with vital teeth. literature review shows that the lpc is more prevalent in adults in the 5th - 7th decades, with mean age of 52 years, without preference for race or sex. the most frequently reported location of lpc is the mandibular premolar area, followed by the anterior region of maxilla (table 1) [5,6,8,11 - 20 ]. in most cases the lpc does not present distinctive clinical symptoms ; the associated teeth are vital, unless secondarily infected. data from clinicopathological studies of lateral periodontal cyst (lpc) the pathogenesis of lpc may be related to the three etiopathological hypotheses : reduced enamel epithelium, remnants of dental lamina and cellular remnants of malassez. the first hypothesis is that the cyst is lined by nonkeratinized epithelium reminiscent of the reduced enamel epithelium which is supported by pcna immunohistochemical expression. the second theory is related to dental lamina remnants, based on the fact that lpc histopathologically presents glycogen - rich clear cells, which is also seen in the dental lamina. the third hypothesis offered that the epithelial remnants of malassez presented in the roots surface, principal location of the lpc, play a role. the diagnosis of lpc should be restricted to cysts that are located in the periodontal side. differentiation between the original lesion and inflammatory cysts and keratocystic odontogenic tumours should be based on clinical, radiographic and pathohistological findings. radiographically, the cyst presents as a well circumscribed round or teardrop - shaped radiolucent area (generally not exceeding 1 cm in diameter) with a radiopaque rim, located laterally to the root of a vital tooth. the periodontal ligament space as a rule is not enlarged and there must not be a communication between the cyst 's cavity and the oral environment [5,11,12,19 - 21 ]. occasionally, lpc may be multicystic, and called as odontogenic botryoid cyst due to macro- and microscopic features resemble to " bunch of grapes " (from the greek word " botrios "). other interradicular radiolucencies must be distinguished from the lpc : anatomic radiolucencies, such as the mental foramen, maxillary sinus and the nutrient canals ; cyst of pulpal origin, other cysts of the jaws, odontomas and other tumours. it may resemble a cyst that develops laterally through a side channel accessory in a non vital tooth. in a retrospective study of injuries of no endodontic origin, 3.8% cases of lpc responsible for various treatments without success the histopathology revealed that lpc is a developmental cyst characterized by a thin layer of nonkeratinized epithelium with a thickness of 1 - 5 mm, which resemble the reduced enamel epithelium. nonkeratinized squamous epithelium is composed of 1 - 5 layers of cells displaying a palisade distribution. the epithelium lining can be rich in epithelial plaques composed of the clear fusiform cells rich in glycogen. some areas of the epithelial thickening, referred to as plaques or theca, are commonly found, and the connective tissue subjacent to the epithelium exhibits a zone of hyalinization. inflammation is not a feature and the walls of the cyst consist of mature collagen fibrous tissue. however, it is possible to observe the histopathological variant of lpc - botryoid cyst, that should receive a greater attention considering the rate of recurrence and unusual presentation. the botryoid cyst represents a histopathological variant which presents with multilocular cystic " grape - like " appearance in the bone. histopathological findings shows multiple cystic spaces lined by nonkeratinized stratified squamous epithelium [14,24 - 26 ]. the mobility of mandibular lateral incisor and canine reported by the patient can be justified by the cyst growth. however, lpc does not reach proportions that are larger than 1 cm while cysts of inflammatory origin tend to grow continuously. in cases of a vital tooth, lpc can still be clinically confused with cysts that develop in inflammatory processes in cases of advanced periodontal disease, where the presence of periodontal inflammation stimulates epithelial proliferation. in most cases the differential diagnosis must be established with radicular cysts, in a view of their high frequency. these lesions are characterized by necrosis of the affected tooth, as a result of which vitality testing proves negative. follicular or dentigerous cysts are always associated to an impacted tooth (particularly a lower third molar), while primordial cysts are mostly located in the ascending mandibular ramus. authors suggest the investigation of the possibility of lpc causing isolated bone defects. it is interesting to know that the soft tissue variant of lpc corresponds to gingival cyst of the adult. adult gingival cysts present the same histogenesis, location and clinical features as lpcs, though the rests of odontogenic epithelium appear in the soft tissues - not in bone as in the case of lpcs. as a result finally, the importance of diagnosis is especially related to the differential diagnosis with keratocystic odontogenic tumour due to its aggressive and infiltrative growth leading to high recurrence rates which requires a more invasive treatment. the keratocystic odontogenic tumour is one of the most aggressive odontogenic tumours due to its relatively high recurrence rate, relatively fast growth, and the tendency to invade adjacent tissues ; it has been reported that it can penetrate even the skull base. scharfetter. demonstrated both : slowly and rapidly proliferating areas in different parts of the keratocystic odontogenic tumour epithelium and the connective tissue wall. he suggested that the invasive growth of keratocystic odontogenic tumour probably resulted from active growth of the connective tissue wall. other possible explanations for its high recurrence are increased fibrinolytic activity in the cyst wall, increased mitotic activity, epithelial proliferation in the connective tissue, and a residual dental lamina with subsequent new cyst formation. the bone resorption by the cyst is mediated by activation of osteoclast - like cells and/or biologically active collagenases. the enucleation is the treatment for lpc, which can often be done without injury to adjacent teeth. the recurrence is not common ; it has been reported with the botryoid variant. the lateral periodontal cyst can be considered in the differential diagnosis when a radioloucent lesion appears adjacent to the roots of vital teeth. the treatment of choice is surgical removal and subsequent histological evaluation to confirm the diagnosis.	abstractbackgroundthe lateral periodontal cyst is considered a developmental odontogenic cyst with unusual occurrence. in most cases it is preliminary diagnosed as a radiographic finding, presenting as well circumscribed or as a round or teardrop - shaped radiolucent area. due to its location it can easily be misdiagnosed as a lesion of endodontic origin. final diagnosis should be based on histopatological examination. the purpose of this paper is to report a classic case of lateral periodontal cyst located in the anterior region of mandible and to review the relevant literature which describes the clinical, radiological and histopathological features of lateral periodontal cysts.methodsa 50 years female patient complained of an asymptomatic gingival swelling in the region between the left mandibular lateral incisor and canine. radiographic examination revealed a well circumscribed radiolucency with approximately 0.5 cm diameter with a radiopaque margin between the roots of the left mandibular lateral incisor and canine. the adjacent teeth had vital pulp.resultsa total enucleation of the lesion was performed, and intraoperative examination showed a single lesion with no communication between the cyst 's cavity and the oral environment. histological examination revealed that the lesion was " lateral periodontal cyst of developmental origin ". there was no recurrence or complications for 24 months follow-up.conclusionsthe lateral periodontal cyst can be considered in the differential diagnosis when a radioloucent lesion appears adjacent to the roots of vital teeth. the treatment of choice is surgical removal and subsequent histological evaluation to confirm the diagnosis. relapses are infrequent.
acquired renal displacements, due to enlargement of adjacent viscera, are less frequent than congenital renal ectopia / malrotation, but have been described. occasionally, these displacements are associated with kidney rotation, more commonly on the right side due to space constraints. here, we report a case of transient right kidney rotation in a patient with a non - ptotic kidney, due to self - limited hepatic congestion and renal enlargement secondary to acute pulmonary thromboembolism. to the best of our knowledge, rapid development and reversibility of acquired kidney rotation secondary to acute pulmonary thromboembolism has not been discussed in the literature. a 43-year - old woman with a prior history of an enterocutaneous fistula, who had required multiple abdominal surgeries, including bowel resections and prior pulmonary embolism, presented to the emergency department with recurrent enterocutaneous fistula and synchronous acute pulmonary embolism. ct scan of the abdomen and pelvis on admission demonstrated rotation and mild inferior displacement of the right kidney, with the long axis of the kidney lying in the horizontal plane [figure 1a and b ]. this was a new finding compared with multiple prior ct scans available within the last 2 years in which the kidneys were normally oriented parallel to the psoas muscles ct pulmonary angiography performed synchronously for syncope and pulmonary decompensation demonstrated a segmental pulmonary embolism and right heart strain, including reflux of contrast material into the inferior vena cava, interventricular septal bowing, enlarged main pulmonary artery (34 mm), and right ventricular dilatation (rv / lv ratio of 1.8) [figure 1c f ]. the most recent ct scan done 45 days prior to this admission is shown in figure 1 g and h. 43-year - old woman presented with recurrent enterocutaneous fistula and synchronous acute pulmonary embolism, and was subsequently noted to have acquired, transient, rotated right kidney. (a) axial contrast - enhanced computed tomography (cect) scan of the abdomen at presentation (day 0) shows rotation of the enlarged right kidney (arrow) around its short axis into the horizontal plane. (b) coronal cect scan of the abdomen at day 0 shows hepatomegaly (double head arrow) with rotation of the enlarged right kidney (arrow head) around its short axis into the horizontal plane. (c) axial cect scan of the chest at day 0 shows enlarged main pulmonary artery (double head arrow) measuring 34 mm. (d) axial cect scan of the chest at day 0 shows segmental embolus (arrow) in the anterior branch of the right upper lobe pulmonary artery. (e) axial cect scan of the chest at day 0 shows dilated right ventricle (double head arrow) with rv / lv ratio of 1.8. (f) axial cect scan of the liver at day 0 shows reflux of the contrast material into the inferior vena cava (arrow) and hepatic veins (arrow head). (g) axial cect scan of the abdomen 45 days before presentation (day 45) shows normal orientation of the normal - sized right kidney (arrow). (h) coronal cect scan of the abdomen at day -45 shows normal orientation of the normal - sized right kidney (arrow head) and normal liver (double head arrow) size. follow - up imaging done after 10 days demonstrated normalization of the right kidney axis, position, and size, as well as decrease in hepatomegaly [figure 2a and b ]. calculation of liver and kidney volumes from ct scans done 45 days before presentation, on the day of admission, and 10 days following admission, using the method described by dello. repeat ct pulmonary angiogram [figure 2c and d ] performed 10 days after presentation showed normalization of the thoracic changes including normal size of main pulmonary artery (25 mm) and normal right ventricular size (rv / lv ratio of 0.86). nephroptosis, which is a condition in which the kidney descends more than two vertebral bodies (or > 5 cm) during a position change from supine to upright, was excluded by ultrasound examination of the right kidney which failed to deviate significantly when the patient changed from supine to upright position. 43-year - old woman presented with recurrent enterocutaneous fistula and synchronous acute pulmonary embolism, and was subsequently noted to have acquired, transient, rotated right kidney. (a) axial contrast - enhanced computed tomography (cect) scan of the abdomen 10 days after presentation (day 10) shows return of the right kidney (arrow) to its normal axis. (b) axial cect scan of the chest at day 10 shows normal - sized main pulmonary artery (double head arrow) measuring 25 mm. (c) coronal cect scan of the abdomen at day 10 shows improvement in hepatomegaly (double head arrow) with return of the right kidney (arrow head) to its normal axis. (d) cect scan of the chest at day 10 shows normal - sized right ventricle (double head arrow) with rv / lv ratio of 0.86. 43-year - old woman presented with recurrent enterocutaneous fistula and synchronous acute pulmonary embolism, and was subsequently noted to have acquired, transient, rotated right kidney. liver and kidney volumes were calculated 45 days before presentation (day 45), at presentation (day 0), and 10 days after presentation (day 10). the volume of the liver was approximately 1390, 2340, and 1944 cm on days 45, 0, and 10, respectively. the corresponding values for the right kidney were 141, 168, and 142 cm, respectively. acquired displacement of the kidney is usually an incidental finding, which may be due to enlarged adjacent organs such as liver, gallbladder, spleen, or adrenal gland. it also occurs in the setting of peritoneal or retroperitoneal masses such as abscesses, hematomas, or neoplasms. this finding is more commonly associated with kidney rotation on the right side than on the left, due to greater space limitations. when caused by hepatomegaly, acquired kidney rotation is usually due to a longstanding process in which the liver enlargement is massive. to the best of our knowledge, rapid development and reversibility of acquired kidney rotation in the absence of nephroptosis has not been discussed in the literature. here, we report a case of transient kidney rotation which occurred over a short period of time secondary to passive hepatic congestion related to an episode of acute pulmonary embolism and resolved promptly within 10 days after the resolution of the right heart strain. hepatic ct volumetry demonstrated a 68% increase in the liver volume at the time of the pulmonary embolism, which may, in part, be due to aggressive hydration after the syncopal episode. however, a few days later, when the right kidney was shown to have returned to its normal anatomical axis and position, the liver volume only decreased by 17%, demonstrating that even small changes in the hepatic size may lead to kidney rotation. a second factor which has been the focus of less attention in the literature is the effect of renal enlargement itself. in the present case, the volume of the kidney increased by 18% at the time of pulmonary thromboembolism and returned to its normal value within 10 days. therefore, it appears that passive congestion and enlargement of the kidney per se can contribute to renal displacement and/or rotation. it has been reported that patients with ptotic, ectopic, or malrotated kidneys may develop vascular or ureteral kinking which can cause a spectrum of renal pathologies including hydronephrosis, pyelitis, renal ischemia, and hypertension. while this did not occur in this patient, the potential exists and it is important for radiologists to be aware of this unusual and interesting entity. we report a case of an acquired, transient, rotated kidney in a patient with acute pulmonary embolism secondary to transient hepatomegaly and passive renal congestion that resulted from right heart strain. to the best of our knowledge, rapid development and reversibility of acquired kidney rotation secondary to acute pulmonary thromboembolism has not been discussed in the literature.	we present a case of an acquired, transient, rotated right kidney in a 43-year - old woman with an enterocutaneous fistula who presented with acute pulmonary embolism. this non - ptotic rotated kidney returned to its normal orientation within 10 days. we postulate that this transient kidney rotation is due to transient hepatomegaly and passive renal congestion secondary to pulmonary embolism. while in this patient there were no untoward sequelae, it has been reported that ureteral obstruction or vascular occlusion can occur in patients with ptotic and malrotated kidneys, and radiologists, therefore, should be aware of this unusual occurrence and the potential complications.
the increased application of self - constructed labview - based chemical virtual instruments (vis) is due to their flexibility and ability to satisfy all the specific user requirements combined with the simplicity of the construction. many configurations of labview - based vi have been reported until now corresponding to their specific chemical application defined by the user needs. described a vi system based on labview 8.0 for ion analyzer which can measure and analyze ion concentrations in solution, comprising a high input impedance voltmeter (widely used in measuring the em generated by ion selective electrode), a homemade conditioning circuit, data acquisition board, and a computer. when applied to determine the reaction rate constant by px, it achieved live acquiring, real - time displaying, automatic processing of testing data, generating a report of results, and other functions. this method simplifies the experimental operation, avoids complicated procedures of manual data processing and personal error, and improves veracity and repeatability of the experiment results. wang. reported a labview - based chemical virtual instrument (vi) for temperatures and pressures measurement. by selecting hardware modules, such as the pci - daq card or serial port method, and the software modules, different kinds of sensors can be used for creating different chemical instruments allowing extremely flexible solutions for automatic measurements in the physical chemistry research. developed a labview - based software for the automation of a sequential injection analysis instrument for the determination of morphine. the detection is based on chemiluminescence reaction with acidic potassium permanganate in the presence of sodium polyphosphate. the authors reported excellent analytical characteristics of the developed labview - based software system to be achieved : the precision of the determination was 0.7% measured by the relative standard deviation for five replicate measurements of morphine standard, and the limit of detection was determined to be as low as 5 10 m. bowie. reported a flow - injection- (fi-) based instrument under labview control for iron monitoring in marine waters the instrument incorporates a miniature, low - power photomultiplier tube (pmt), and a number of electric and solenoid actuated microvalves and peristaltic pumps. the software allows full control of all flow injection components and the processing of the data from pmt. the optimized system is capable of 20 injections per hour, including preconcentration and wash steps. the analytical characteristics of the developed labview - based system are reported to be as follows : a detection limit of 21 pm at linear range of 212000 pm with a 60-second sample load time and average precision between replicate fi peaks of 5.9 3.2% (n = 4) over the linear range. the flexibility of the vis allows their application practically in any branch of the chemical technology. for example, the quality control of the conversion coating on aluminum alloys requires the determination of the pits number appearing on the 3 10 inches control specimens after their testing in saline chamber at extreme conditions : high temperature, high relative humidity, and high saline concentration, according to the standard astm b117. according to this standard, the number of the appearing pits is the measure of the corrosion resistance of the protective coating. the pits counting however actually made by simple specimen observation results in subjective errors due to the bad distinction of the pits from some pits - like simple stains and hence false results about the corrosion resistance of the conversion coatings. that is why a rapid and subjective error - free method for pits counting is necessary. thus, the purpose of the present work is to develop such a vi and to test it on real specimens for express and objective pits counting. such a vi must determine the pit centers coordinates, pit areas, their traverse lengths, and the densities using blobs analysis resulting in a map file which can be used further by a svet system [57 ] to perform a rapid (one pass) true / false check of the probable pit containing zones only, without scanning of the entire specimen. a vi fulfilling all these requirements using a particle analysis based on blobs analysis included in imaq libraries for labview 2010 called localized corrosion image analyzer (lcia) was developed and described in the present paper. its hardware and software are presented together with some application for pit recognition on real metal samples. the specimen optical scan performed by a digital microscope connected to a pc yields a database file (a map file) containing the coordinates of all the surface defects similar to pits, not only the ones caused by corrosion. image analysis performed by labview 2010 was applied for preliminary image recognition and distinction of the pits. true / false svet test application allowing the distinction of the true corroded (pit containing) zones from the corroded - like ones to be used further from a svet system for corrosion rate determination. the true / false test is a rapid single linear svet scan over the centers of the probable zones in which coordinates are saved in the created by the vi lcia map file. the simple linear svet scan will result in a specific and easy recognizable peaks contained curve in coordinates : current intensity / coordinate. the scanning vibrating electrode technique (svet) which was developed for biological applications was adapted later by isaacs [6, 7 ] for corrosion studies of pitting, intergranular, and galvanic corrosion. the determination of the current distribution above the metal surface measuring the potential gradient (voltage drop) proportional to the ionic current density e = (i1 i2)r that appears between the two levels of the electrode vibration (from 1 to 100 m) above the studied surface. svet is a further development of the scanning reference electrode technique (sret) [8, 9 ] but provides higher sensitivity due to a.c. signal measurement allowing obtaining a higher signal to noise (s / n) ratio due to the better noise suppression. for example, the minimal voltage drops levels measurable by sret are about 200 v [10, 11 ], while 5 v can be achieved by svet [1214 ]. the labview - based vi subject of the present paper employs the first main point of the approach developed by the authors based on the following two main points : computer vision application for video inspection by digital microscope of the surface of interest and database (map file) creation of the probable pits containing areas;rapid true / false test by single linear svet scan for real pits distinction followed by a map file actualization. computer vision application for video inspection by digital microscope of the surface of interest and database (map file) creation of the probable pits containing areas ; rapid true / false test by single linear svet scan for real pits distinction followed by a map file actualization. additionally, a complete svet scanning of the recognized real pits areas can be performed if the corrosion rate determination is necessary. the vi performing the video inspection and the creation of the map file only is the subject of the present work, while the svet vi is the subject of another publication. three main devices were involved in the system hardware configuration : a video inspection mighty scope near infrared 10x200x, 1.3 mp, 1/4 cmos color, 6 led light type operating at 850 nm, usb 2.0 interfaced digital microscope with a homemade lineal stage focus controlling mechanism in the range from 8.5 mm to 112 mm, with stepper motor npm pf35 - 24c1, a homemade svet device with x - y stages, stepper motors vexta model px245 m-01a, and a personal computer dell optiplex 7010 core i7 cpu @ 3.40 ghz, windows 8 pro operating system as illustrated in figure 1. the optical inspection by a digital microscope allows the capturing of full images from 4 mm to 1700 mm (optical zoom in the range from 10x to 200x), respectively, of the studied surface with a resolution of 1600 to 1200 effective pixels at shot speed user controllable from 1 to 1/1000 sec. the focus adjusting mechanism (y stage, figure 1) was driven by a stepper motor controlled by the lcia which controls also the real time image capturing and its transfer through the usb interface. the white balance was carried out automatically employing the integrated 6 white led light ring around the lenses. the homemade svet device which uses the map file produced by the vi subject of the present work consists of a vibrating electrode with edge diameter of approximately 1 m, 100 mv p - p, and frequency of the vibration of 60 hz at amplitude of the displacement adjustable in the range between 1 and 100 m over the specimen surface. the vibrating electrode was mounted on x - y linear stages mechanism providing 5 m / step resolution. the svet electrode x - y displacement and vibration were controlled by a separate vi instrument running its own software and communicating with the lcia, using the map file containing the pits coordinates previously obtained with the lcia. the svet analog signal was amplified by a controlled gain high input impedance instrumentation preamplifier (10 ohm/100 fa), and after its digitalizing by national instruments, model usb 6009 data acquisition system (daq) digital signal was transferred to the pc through the usb port and processed by its vi lock - in amplifier labview tool. the lcia focuses, captures the image of the specimen surface, and analyzes it creating a database file in real time containing the exact coordinates, area, and transverse length of probable pits. this file is employed by a svet system with its own control software, performing the true / false test to distinguish the true pits coordinates. in true case, a complete svet scan of the probable area can be performed and the corrosion rate be determined, if preliminary chosen by the operator in the interface screen. in false case, the svet electrode goes to the next probable area up to the last using optimized trajectory. in both cases, after the true / false test performance, an updating of the already existing database file takes place. the flow diagram of vi lcia system shown in figure 2 is divided in two main subvirtual instruments : image digitizing sub - vi and image analyzing sub - vi, both executed to get the complete pits location information. the image digitizing sub - vi creates the sample 's digital image by making a preview, focusing, image capturing, histogram display, and focus verification entirely synchronized in real time with the image analyzing sub - vi. stacked sequence 1 structure where the image digitizing sub - vi is executed for frame 0 of stacked sequence 1 and the image analysis sub - vi is executed for frame 1 of stacked sequence 1 (see figure 2). the image digitizing sub - vi is located in frame 0 of the stacked sequence 2 inside the stacked sequence 1 shown in figure 3, where the functions : focus and image preview, are performed simultaneously within a while loop execution control. the focus has two nested case structures. when the main case structure is true, the focus starts by the execution of focus section sub - vi through a case true substructure ; this process calls up the node for writing via usb to control the driver of stepper motor mounted on the focus mechanism ; it focuses by using a for cycle and a structure type stacked sequence (programming shown on figure 3). when the main case structure is false, however, it calls up through the usb port the writing node which indicates the motor driver to de - energize the motor, and by this way, the focusing process stops. the previously mentioned procedure sets the forward or backward sequence to control the execution time, the speed of the focus mechanism stepper motor, and the focus termination through the control driver. the image preview is made by using the labview ni imaqdx libraries, tools that allow the reading of camera or microscope through a usb port. this job is performed within an event structure (see figure 3) and allows simultaneous real - time image observation and focusing. the image preview is taken by the application of the labview ni imaqdx tool allowing automatic detection and camera(s) selection by the user via the usb port. this sub - vi is implemented outside the while loop and inside the event structure shown in figure 3. once the capture button, located on the sub - vi front panel is pressed, the frame 0 of stack sequence 2 terminates, and the frame 2 starts automatically, capturing the image and saving it in.jpg format file (the programming is shown in figure 4). after saving the generated file the sub - vi content generates the corresponding histogram for the image capturing. figure 5 shows the user graphic interface (the front panel of vi lcia for the image digitizing), where the corroded surface image already captured is displayed together with the corresponding histogram. according to the histogram values the user decides to keep the image for further analysis or to delete it and run the process again. in the image analysis applied by vi lcia to metal samples for pitting corrosion determination, described on this document, the blob analysis of imaq library from labview 2010 this technique involves several vision operations and analytical procedures, which detect within the image any 2d object which could be a potential pit located on the studied corroded metal surface. the image processing generates blobs and then works with them to calculate the area and perimeter of the pit, and the number of the generated blobs is an important characteristic for pits distinguishing. blob (binary large object) is a group of interconnected pixels that have the same logic state and the same light intensity. the basic structure of an image recognition vi by the application of blob analysis in imaq labview consists of image acquisition, histogram calculation, thresholding, and blob 's filtering and analysis. the image recognition vi for pitting corrosion application has all these components as well as the morphology and particle labeling to improve the shape and definition of pits under investigation. the programming diagram of the image analysis sub - vi is shown in figure 6. by means of the icons imaq create and imaq read file the precaptured images are acquired into the sub - vi for digitizing. after that, the color threshold process is performed to turn the image into binary format in the first section. once the threshold process is done, the desired morphology of pitting is predefined by imaq morphology, and then a filtering process is performed by imaq particle filter which can be used to clean up the image deleting the nondesired particles defined as noise. the pitting spots in the already clean image are labeled and highlighted in different colors, by imaq label, so they can be easily distinguished. finally, the pitting spots which are pixels are counted to be converted in real metric units by imaq particle analysis, and saved together with the lcia generated parameters as : pit location, area, and transverse length. once the blob analysis is performed, the system proceeds to convert pixels to metric units for all the parameters determined by the blob analysis (program process shown in figure 5). the conversion to square millimeters of the area is performed by the subarray from array subset area ; the conversion to millimeters of the length is performed by the subarray from array subset length, and finally the location by coordinates in the subarrays (x, y) performed by array subset for coordinate x and coordinate y, respectively. the system vi lcia was applied for image recognition in localized corrosion studies of aluminum alloys aa6061-t3 specimens determining the number, density, and coordinates of probable pits areas together with their dimensions. then, the created database file was employed for real - time scanning of the affected by the corrosion areas only employing svet improving thus the scanning time more than ten times compared with the time for complete svet scan of the entire surface. in figure 7(a) left is shown the microscope image of a carbon steel uns g10180 sample obtained with vi lcia application. there, one of the pits is highlighted with a circle as an example, and in the right part of the same figure its identification by vi lcia is shown determining its area to be 0.041 mm and its transversal length to be 1.76 0.94 mm as displayed in the graphic interface of the image analysis sub - vi. these values fit very well with those obtained by the svet device and the amf microscope application. the total time for the svet scan of the 25 25 mm specimen with the lcia application was found to be about 4% only of the total time required for complete svet scan of the same specimen achieving an acceleration of about 25 times. a virtual instrumentation system called vi lcia based on labview 2010 was developed allowing rapid determination of the pits number on large metal specimens. the vi lcia controls synchronously the focus adjustment and the image taking by a digital microscope and the image analysis, resulting in detection and the mapping of the probable pits containing zones on the corroded metal specimen with their traverse length and density using blobs analysis. the created map is further used by a homemade svet vi system with its own labview 2010 software to control a rapid performing (one pass scan) true / false check of the probable pits containing zones and also is able to perform a complete svet scan of the already recognized true zones to determine the corrosion rate.	a virtual instrumentation (vi) system called vi localized corrosion image analyzer (lcia) based on labview 2010 was developed allowing rapid automatic and subjective error - free determination of the pits number on large sized corroded specimens. the vi lcia controls synchronously the digital microscope image taking and its analysis, finally resulting in a map file containing the coordinates of the detected probable pits containing zones on the investigated specimen. the pits area, traverse length, and density are also determined by the vi using binary large objects (blobs) analysis. the resulting map file can be used further by a scanning vibrating electrode technique (svet) system for rapid (one pass) true / false svet check of the probable zones only passing through the pit 's centers avoiding thus the entire specimen scan. a complete svet scan over the already proved true zones could determine the corrosion rate in any of the zones.
	abstractthe connection of embryonic stem cell technology and developmental biology provides valuable tools to decipher the mechanisms underlying human brain development and diseases, especially among neuronal populations, that are not readily available in primary cultures. it is obviously the case of neurons forming the human cerebral cortex. in the images that are presented, the neurons were generated in vitro from human embryonic stem cells via forebrain - like progenitors. maintained in culture for prolonged time, they acquired a mainly glutamatergic phenotype and morphological characteristics of cortical pyramidal neurons, including dendritic spines, and formed spectacular networks.
paraganglioma is a rare neuroendocrine neoplasm that may develop at various body sites like abdomen, thorax, head and neck. majority of paragangliomas develop in abdomen while mediastinal paraganglioma is a rare mediastinal tumor.12 posterior mediastinum is the commonest site for mediastinal paraganglioma while anterior mediastinal localization is unusual.13 most paraganglioma are asymptomatic and present as painless mass. endoscopic ultrasound (eus) features of retroperitoneal paraganglioma have been described4 while that of mediastinal paraganglioma have not been described. in this report we describe the eus features of mediastinal paraganglioma in a patient who presented with cough and chest pain without any neuroendocrinal symptoms. a fifty - six year old female presented with one month history of chest pain and cough. the patient was not hypertensive and did not give any history related to neuroendocrinal hormone over secretion. the mass was inverted triangular shape with bulging peripheries, situated between aorta and left pulmonary artery. the mass was hypervascular in the center and the interface between the mass and vessels described was intact and there was no lymphadenopathy (fig. 1 - 3). eus - guided fine needle aspiration (eus - fna) was done using a 22-g needle (echotip, cook corporation). slides were prepared and the material was air dried as well as alcohol fixed before sending for cytopathological evaluation (fig. 4, 5). good material was obtained on eus - fna, but three experienced cytopathologists gave three different reports ; final diagnosis of paraganglioma was only made on thoracoscopic biopsy and immunohistochemistry. mediastinal paraganglioma is a rare mediastinal tumor.12 it constituted 0.3% of all mediastinal tumors in the series described by cesar.1 while no case of mediastinal paraganglioma was found out of 57 eus - fna done for mediastinal masses by zeppa.2 majority of thoracic paragangliomas occur in posterior mediastinum. out of 16 cases of thoracic paragangliomas reported by cesar paragangliomas arise from parasympathetic or sympathetic ganglia located in ap window or posterior mediastinum.34 diagnosis of asymptomatic paraganglioma on any imaging modality is difficult. on fna cytology, it is difficult to give final diagnosis.4 in our patient also the tumor could be finally diagnosed only upon thoracoscopic biopsy and immunohistochemistry and the tumor was positive for chromogranin, synaptophysin and neuron specific enolase. eus features of mediastinal paraganglioma are not described while only in one case eus features of retroperitoneal paraganglioma are reported.2 the situations in retroperitoneum and mediastinum are different. retroperitoneum is a potential big space where tumor can grow freely to any size and can assume any shape while there is little space in mediastinum for tumor to grow and tumor may assume a particular shape. in the present case, there were certain features which were highly suggestive of paraganglioma. it is inverted triangular shape with base of the triangle lying superiorly, because there is sufficient space in ap window where tumor can grow, and apex lying inferiorly, because the tumor has to grow in between the vessels. besides, there were other features such as rich vascularity of the tumor, bulging peripheries, no invasion of vessels and absence of lymphadenopathy. we analyzed our 50 cases of various mass lesions involving ap window and subcarinal space, and in none of the cases such typical eus features were seen. growth in between the vessels without invasion of vessels was a typical feature of benign paraganglioma. when such appearance is seen on eus imaging, diagnosis of paraganglioma is quite likely and eus - fna may be risky and may not provide final diagnosis.	endoscopic ultrasound (eus) features of mediastinal paraganglioma have not been described. in this paper, we report a female patient presented with cough and chest pain without any neuroendocrinal symptoms. final diagnosis of mediastinal paraganglioma was made on thoracoscopic biopsy and immunohistochemistry after eus - guided fine needle aspiration. eus features of mediastinal paraganglioma are described.
diabetes mellitus (dm) is a serious public health concern that causes illness, disability, and death throughout the world. clinical diabetes therapy requires precise monitoring and maintaining of blood glucose levels as close to normal as possible to reduce the risk of emergency complications such as the retinopathy, nephropathy, and hypo- and hyperglycemia [13 ]. to date, existing sensing techniques have been demonstrated, such as glucose - based noninvasive glucose sensors and enzyme - based biosensors ; however, impaired responses and unpredictable drift make these unsuitable for long - term therapeutic practice. therefore, the development of new diagnostic tools for reliable glycemic control with sufficient sensitivity and selectivity is of clinical interest. in order to solve these problems, microcantilever - based sensors have been proposed, which have high sensitivity, low detection limits, and broad application in the fields of chemistry, biotechnology, detection, and medical science [4, 5 ]. this sensing system has significant advantages over other sensor technologies, such as high surface - to - volume ratio, fast response time, and low cost of fabrication, and further, has proven effective in dna hybridization and, biomolecular recognition. moreover, attogram - level mass detection sensitivities have been achieved using cantilever - based sensing technology. microcantilevers have been used as force probes in atomic force microscopy (afm) and for high - resolution imaging to examine the topography and mechanical properties of biological samples without labels or treatment. further, the cantilever may be functionalized with various probe molecules (e.g., dna, proteins, and antibodies) and used as a specific biosensor for target molecules (e.g., dna and antigens). this sensing system can be further specialized to act as a functional biosensor, whereby the resonance frequency of the cantilever shifts with mass loading when biomolecules absorb onto a treated tip. in the present study, we have used microcantilevers (via the vibration method) and measured the resonance frequency shifts for glucose recognition and mass detection. we chemically modified the silicon afm microcantilevers with aminopropylsilatrane (aps), followed by adsorption of concanavalin a (con a) onto the aps - modified probe. glucose - specific lectin con a has a strong affinity for glucose and has been used as a glucose monitoring sensor [9, 10 ]. we used the con a / aps - modified probe to detect glucose and galactose in solution and then compared the frequency shifts to determine which type of sugar attached to the con a. we demonstrate that the afm - based microcantilever method of glucose detection offers a powerful technology which can achieve attogram mass sensitivity in clinical microbiology systems for management of glucose levels of dm patients. an aminopropylsilatrane (aps) solution was prepared using a synthetic method reported earlier [11, 12 ], and a stock sample of 50 mm aps in milli - q reagent grade (type i) water (18.2 m-cm at 25c) was prepared and stored at 4c.. con a, from canavalia ensiformis (jack bean) (type vi, lyophilized power, mw 26.5 kda), was purchased from sigma - aldrich (usa) and was used to prepare a 1000 ppm con a solution. d(+) glucose (dextrose anhydrous) was purchased from showa, and was used to prepare a 1% glucose solution. d(+) galactose was purchased from sigma - aldrich (usa) and was used to prepare a 1% galactose solution. throughout the experiments, milli - q reagent grade (type i) water (18.2 m-cm at 25c) was used. all cantilever probes were plasma cleaned (harrick scientific products, inc.) for 2 min using dry air as the reactive gas to increase the oh concentration at the surface [15, 16 ]. arrow force modulation mode probes (arrow - fmr) were purchased from nanoworld ag (switzerland). the cantilevers were 240 m long, 35 m wide, had a triangular free end, and tetrahedral tip shape with a height of 1015 m with a measured normal spring constant of 2.8 n / m (thermal method) [17, 18 ]. to prepare the probes for glucose detection, plasma cleaned cantilevers were immersed in a diluted aps solution (volume ratio of aps : water = 1 : 300) for 30 min, removed, rinsed thoroughly with milli - q water, and then dried under a stream of pure nitrogen gas. following this procedure, alkylsilane molecules were chemically attached to the afm tip, much like self - assembled monolayers on silicon substrates from our previous study. we then put the aps - coated cantilever into a 1000 ppm con a solution for 15 min, removed and rinsed thoroughly with milli - q water, and then dried under a stream of pure nitrogen gas. finally, the aps / con a modify cantilever was immersed in a 1% glucose solution for 15 min, rinsed thoroughly with milli - q water, and dried under a stream of pure nitrogen gas. p - type / boron dopant ; resistivity 110 cm) were used for complimentary experiments where the si substrates were treated with aps and con a and then exposed to glucose or galactose. changes in surface roughness and topography, as measured by afm, were related to glucose or galactose adsorption. topographic images were obtained using an afm (mfp-3d, asylum research, santa barbara, ca, usa) operating in ac mode under ambient conditions. a silicon cantilever (nano world ag, arrow - fmr) with a nominal spring constant of 2.8 n / m was used for all images, with a scan rate of 1.0 hz and image resolution of 512 512 pixels. figure 1 is a cartoon depicting the process of chemically functional afm tip to attach the sample. as shown in figure 2(a) (), the aps - modified cantilever had a resonance frequency of 65,948.8 5.0 hz in air. con a was chemically attached to the cantilever by immersing the cantilever into the con a (1000 ppm) solution for 15 mins and then immersing the probe into ultrapure water to rinse off the unbonded residues. figure 2(a) () shows that the resonance frequency after the addition of con a was 65,876.9 5.5 hz. the resonance frequency of the cantilever is given by (1)f=12km, where k is the spring constant and m is the effective mass of a cantilever. the measured downshift in the cantilever resonance frequency confirms that the con a molecules have attached to the tip, and these molecules have increased the cantilever effective mass. the frequency shift obtained using (1) was used to estimate an effective mass increase of 9.13 pg, which corresponds to 3.45 10 moles of con a attached to the cantilever. the same probe was then used to detect glucose by incubating the probe in the prepared glucose solution and then rinsing in ultrapure water to remove the unbonded residues and drying. immediately following glucose exposure, we observed that the cantilever resonance frequency had shifted lower to 65,837.8 2.8 hz as shown in figure 2(a) (). using the same calculation, we estimated an effective mass increase of about 4.98 pg, which corresponds to the addition of 2.76 10 moles of glucose to the probe. as a negative control, we ran a similar experiment using galactose rather than glucose as the analyte. although the molecular formulas for glucose and galactose are identical, they are structural isomers, where the difference is the turning up (or down) of the hydroxyl group on one of the carbons. using the same process, a coated con a - modified probe was immersed into the galactose solution for 10 min and then immersed in ultrapure water to rinse off the unbonded residues. as shown in figure 2(b) (), the aps - modified cantilever exhibited a resonance frequency of 67,709.4 6.0 hz. following con a attachment, the resonance frequency shifted to 67675.5 3.5 hz (), which corresponded to a mass increase of 4.6 pg, or 1.73 10 moles of con a attached to the cantilever. after incubation in the galactose solution, the measured resonance frequency was 67,669.9 3.0 hz () which was within the measurement error of the con a (only) measurement () and indicates that galactose did not attach to the con a - modified afm probe. to verify the detection step of our method, we ran a similar experiment on a silicon wafer substrate and monitored morphology changes using afm topographical imaging. the same sample preparation procedures were used for the silicon wafer substrates as were used for the silicon microcantilevers. figures 3(a) and 3(d) show afm images of the aps - modified surface morphology. the surfaces were very flat, with root mean square (rms) roughness of 67 and 65 pm, respectively. figures 3(b) and 3(e) show the same substrates after con a was adsorbed onto the aps - modified surfaces. the surface roughness increased for both surfaces to 682 and 675 pm, respectively. the surfaces shown in figures 3(b) and 3(e) were then treated with glucose and galactose, respectively. the surface treated with glucose (figure 3(c)) exhibited an increase in rms roughness to 928 pm and evolved a stepped topography with a network of holes ~1.5 the surface shown in figure 3(f), on the other hand, showed similar topography as that for the con a - modified surface (figure 3(e)) with a negligible change in surface roughness (697 pm). within each of the figure 3 topography images, we plotted line scan profiles to more clearly display the surface topography. the straight red line in each image shows where the cross - section data was obtained. in figures 3(a) and 3(d), the line scan is flat, indicating that the surface is very smooth. figures 3(b) and 3(e) show that the line section over a 1 m range has a peak to valley height of 650 pm. the line section in figure 3(c) shows that the holes have a depth ~1.5 nm. all the line sections are plotted on the same scale, with the scale bar shown in figure 3(a). these results indicate that glucose adsorbed onto the con a - treated surface, while galactose did not. this further confirms the results from our cantilever resonance frequency measurements where glucose (but not galactose) was detected. we have demonstrated a simple and effective method for detecting glucose using a chemically modified afm probe and estimating the change in mass by monitoring the resonance frequency shift of the cantilever. this method may be used to investigate lectin - carbohydrate interactions in the form of carbohydrate and lectin arrays or more broadly to study a wide range of cell - cell and antigen - antibody bonds. with attogram mass sensitivity, this technique shows great promise for the development of frequency detection sensor technology.	efficient maintenance of glucose homeostasis is a major challenge in diabetes therapy, where accurate and reliable glucose level detection is required. though several methods are currently used, these suffer from impaired response and often unpredictable drift, making them unsuitable for long - term therapeutic practice. in this study, we demonstrate a method that uses a functionalized atomic force microscope (afm) cantilever as the sensor for reliable glucose detection with sufficient sensitivity and selectivity for clinical use. we first modified the afm tip with aminopropylsilatrane (aps) and then adsorbed glucose - specific lectin concanavalin a (con a) onto the surface. the con a / aps - modified probes were then used to detect glucose by monitoring shifts in the cantilever resonance frequency. to confirm the molecule - specific interaction, afm topographical images were acquired of identically treated silicon substrates which indicated a specific attachment for glucose - con a and not for galactose - con a. these results demonstrate that by monitoring the frequency shift of the afm cantilever, this sensing system can detect the interaction between con a and glucose, one of the biomolecule recognition processes, and may assist in the detection and mass quantification of glucose for clinical applications with very high sensitivity.
ticks are considered important carriers of pathogenic microorganisms in the northern hemisphere, and ixodes ricinus ticks are associated with a diverse microbiota. borrelia spirochetes are one of the pathogens transmitted to humans by i. ricinus ticks, and borrelia burgdorferi sensu lato (s.l.) represents a bacterial species complex that comprises several borrelia genospecies associated with lyme borreliosis (lb). borrelia burgdorferi sensu stricto (s.s.), borrelia afzelii, and borrelia garinii are causative agents of human lb in norway, and the prevalence of lb is monitored by the norwegian surveillance system for communicable diseases (msis). a fourth these four genospecies have been detected in i. ricinus ticks in northwest norway. during the recent years, several genotyping methods have been established for analysis of borrelia to characterise strains and determine the phylogenetic relationships between strains [711 ]. multilocus sequence typing (mlst) is a molecular typing tool that is used to characterise pathogenic microorganisms, and mlst can be used as a tool for the epidemiological surveillance and tracking of infectious diseases. mlst, as defined by urwin and maiden, is a genotyping tool based on the sequences of housekeeping genes that evolve slowly. the sequences used for mlst are approximately 400500 bp in size and are located throughout the genome to avoid bias. multilocus sequence analysis (mlsa) applies mlst to characterise closely related species using a distance - based procedure for phylogenetic characterisation, and it includes pairwise genetic similarities. mlst / mlsa have been applied in population studies and for analysis of borrelia species in geographic areas and for evolutionary studies and characterisation of new borrelia species. while some mlst / mlsa schemes combine housekeeping genes with hypervariable regions and noncoding loci [911 ], the mlst scheme published in 2008 by margos. is based on housekeeping genes that fulfill the strict criteria defined by urwin and maiden [7, 13 ]. the borrelia mlst scheme is based on amplification, sequencing, and bioinformatic analysis of internal fragments of eight housekeeping genes (clpa, clpx, nifs, pepx, pyrg, rplb, recg, and uvra). recently, studies of phylogenetic relationship of borrelia genospecies using mlsa have resulted in the definition of two new borrelia species, borrelia bavariensis sp. and borrelia kurtenbachii. their findings indicated that, to a degree, strain diversity is influenced by the host. the aim of this study is to utilise mlsa to characterise borrelia strains isolated from i. ricinus ticks collected from the fauna in northwest norway. multilocus sequence analysis of norwegian strains could provide knowledge about the strain diversity among borrelia strains found in this region and describe their evolution compared to european strains. dna from questing i. ricinus ticks collected in 2012 (531 nymphs/71 adults) and 2013 (539 nymphs/64 adults) (figure 1) was isolated, and the dna samples were analysed by qpcr to detect the presence of borrelia genospecies as previously described. a total of 86 samples (79 nymphs/7 adults) were positive for borrelia in 2012, and 89 samples (83 nymphs/8 adults) were positive for borrelia in 2013. a total of 50 samples were amplified across all eight housekeeping genes (clpa, clpx, nifs, pepx, pyrg, recg, rplb, and uvra) and were submitted for sequencing. the amplification of each housekeeping gene was carried out in a 15 l reaction volume containing 7.5 l mastermix 2.0x sybr green (applied biosystems), 0.75 l of each primer (10 m), 3.00 l ddh2o, and 3.0 l template dna. amplification of the eight housekeeping genes (clpa, clpx, nifs, pepx, pyrg, recg, rplb, and uvra) was performed with primers and nested touchdown pcr conditions for clpa, nifs, pepx, pyrg, recg, and uvra as previously described [7, 8 ]. combined with a nested touchdown pcr composed of 1 cycle of denaturation (10 min, 95c), followed by 9 cycles of denaturation (30 sec, 95c), touchdown annealing (30 sec, 58c50c), and extension (1 min, 72c) and 30 cycles of denaturation (30 sec, 95c), annealing (30 sec, 50c), and extension (1 min, 72c) with a final cycle of extension (10 min, 72c). gel electrophoresis was used to confirm the size and approximate concentration of the pcr products for each amplified gene. gel electrophoresis was performed with a 2% tris - acetate edta buffered (tbe) gel followed by staining with gelred (affymetrix, santa clara, usa). the pcr products were purified in a two - step process by exosap - it (affymetrix, santa clara, usa) according to the manufacturers ' protocol. the sequencing reaction was carried out in a 10 l reaction volume containing 1 l big dye terminator 3.1 (applied biosystems, carlsbad, usa), 1 l 5x sequencing buffer (applied biosystems, carlsbad, usa), 0.32 l primer (10 m), 4.68 l ddh2o, and 3 l template dna. amplification was performed in a 2720 thermal cycler (applied biosystems, carlsbad, usa) with 1 cycle of denaturation (6 min, 96c), followed by 25 cycles of denaturation (10 sec, 96c), annealing (5 sec, 50c), and extension (4 min, 60c). finally, 10 l ddh2o was added to each well upon completion of the sequencing reaction, and the samples were stored at 20c until sequencing. the sequences for each housekeeping gene were aligned by the muscle algorithm and concatenated in mega 5.1. the sequences were analysed with blast to confirm the correct borrelia genospecies. each locus and the concatenated sequence of all eight loci the mega 5.1 software was used to draw a distance matrix neighbour - joining tree (bootstrapped 500 iterations) with the concatenated dna sequences. sequences from b. duttonii were added from the blast database to root the neighbour - joining tree. sequences from four reference species from the mlst database were included in the analysis, b. burgdorferi s.s. (b31), b. afzelii (vs461), b. valaisiana (70865b), and b. garinii (20047). to compare allele sequences and sequences types (sts) and to identify identical sequences, the nonredundant database (nrdb) written by warren gish at washington university all known alleles and sts were retrieved from the borrelia mlst database for comparison (http://borrelia.mlst.net/). sts that were not found in the mlst database were submitted to the mlst database for proper numbering. statistical analysis was performed with mega 5.1. the mean pairwise genetic difference (within and between groups) average nonsynonymous substitutions and synonymous substitutions (dn / ds) were calculated using the modified nei - gojobori method (jukes - cantor). the number of polymorphic sites (ps) and the nucleotide diversity () were calculated using tajima 's test of neutrality. the index of association, both standard and classic, was calculated using start2. to compare the relationship between norwegian borrelia strains and european borrelia strains a goeburst minimum spanning tree was generated using phyloviz 1.0 software. a total of 50 strains were sequenced across all eight loci for the present study. a distance matrix neighbour - joining tree (bootstrapped 500 iterations) (figure 2) demonstrated that the 50 strains were separated into 36 sequence types (sts) (table 1) that were not previously found in the mlst database. the neighbour - joining tree formed four deeply branched clusters, and, between the four clusters, there were no shared alleles. all strains cluster together with their respective reference species and contain combinations of alleles that are new entries into the mlst database. the mean pairwise genetic difference confirms the genospecies clustering (figure 3) based on the genospecies threshold (0.017) as defined by margos.. table 2 summarises the characteristics of each of the eight housekeeping genes included in the multilocus sequencing analysis of borrelia strains. the comparative goeburst analysis (level 8 grouping) displays the relationship between the norwegian strains and european strains (figure 4). the goeburst analysis shows that the norwegian borrelia population is the most closely related to the latvian and french populations. a goeburst analysis was performed at the slv (single locus variant) level by dividing the dataset into 32 groups with 2 or more sts and 64 singletons. the largest clonal complex 0 (cc0) has 13 unique sts and represents 33 strains. all of the slvs share the pepx, pyrg, rplb, and uvra alleles. in cc1 (9 sts, 22 strains), the predicted ancestral genotype is st 86, with 8 slvs representing 10 strains ; thus this is the highest ranking ancestral genotype. all slvs have the same clpa, clpx, and rplb alleles. a goeburst analysis conducted at the dlv (double locus variant) level divided the dataset into 54 ccs consisting of 26 groups with 2 or more sts and 28 singletons. a goeburst analysis conducted at the tlv (three loci variant) level divided the dataset into 33 ccs consisting of 21 groups with 2 or more sts and 11 singletons. a level 4 grouping was required for b. valaisiana to be represented by one cc in the goeburst analysis, and a level 5 grouping was required for b. afzelii to be represented by one cc. a level 7 grouping was required for b. garinii and b. burgdorferi s.s. to be represented by one cc. the norwegian strains are spread throughout the goeburst tree and are clustered together with their respective genospecies from other european countries. these four branches contain sts related to the four genospecies, b. garinii, b. afzelii, b. valaisiana, and b. burgdorferi s.s. the highest ranking ancestral genotype, st 86, is a b. garinii strain from latvia. northwest norway is characterised by geographical features such as islands, several deep fjords, and high mountains. the landscape shows signs of overgrowth and little maintenance of the vegetation, thus providing suitable growth conditions for diverse wildlife. the region has mild winters and humid summers, creating good conditions for ixodes ricinus ticks. borrelia spirochetes are maintained in nature by the circulation between the vector and the host, and the wide range of available hosts may influence the diversity among strains of borrelia. borrelia spirochetes have an obligate parasitic lifestyle, and their circulation depends on an interaction between reservoir hosts and vectors. birds are reservoir hosts for b. garinii while b. afzelii are usually associated with rodents [15, 25 ]. evolution and a wide range of determinants that affect reservoir hosts and vectors are likely to influence the diversity among strains differently [23, 24, 26 ]. reservoir hosts and vectors are submitted to a number of factors such as climate, nutrition, and pollution that have different impacts depending on the geographic locations [23, 24 ]. in addition to the influence that reservoir hosts provide, the complex microbial communities associated with i. ricinus ticks also subject borrelia spirochetes to microbial competition and natural selection. these microbial communities are composed of bacteria, endosymbionts, viruses, and protozoa from the tick 's natural habitat and hosts, and, therefore, they vary between different geographical locations [2, 27, 28 ]. multilocus sequence analysis of the borrelia strains found in i. ricinus ticks from this region shows signs of high diversity. the majority of the strains are b. afzelii and b. garinii, the most prevalent genospecies within this region. fifty borrelia strains were separated into 36 sts, and the genetic relationship between the strains was visualised in a neighbour - joining tree. the first deeply branched cluster contains strains identified as b. afzelii, and 29 strains were separated into 22 sts. these 29 b. afzelii strains form a highly structured population consisting of sts that have not been previously described in the mlst database. in 2011, vollmer. demonstrated that there is limited movement of b. afzelii strains between geographic locations and that only two b. afzelii sts have been identified in more than one geographic location [22, 29 ]. b. afzelii strains are described as heterogenic, and a comparative goeburst analysis of strains from scotland and strains from england showed that there is a limited movement of strains between the north and south in the united kingdom [24, 29 ]. b. afzelii is associated with small rodent hosts with a limited movement pattern, thus creating a limited dispersal of b. afzelii strains. all strains included in the present study were collected from four sites within one geographically defined area (figure 1). some of the b. afzelii strains were identified in more than one sample, but they were always within the collection site. the second deeply branched cluster contains four b. valaisiana strains divided into 2 sts that represent new entries into the mlst database. the third deeply branched cluster contains 16 b. garinii strains divided into 11 sts that are new entries into the mlst database. both b. valaisiana and b. garinii are bird - associated genospecies, and previous studies have found that these two borrelia genospecies have sequence types that have been identified in different geographic locations throughout europe. vollmer. reported that b. valaisiana strains are not completely homogenised within europe while b. garinii shows signs of being a mixed population across central europe. migrating birds play an important role in the dispersal of b. garinii strains between geographic locations. studied the prevalence of borrelia infection in 31 species of migrating birds and 6 species of resident birds in southern norway. studies also suggest that b. garinii strains have the ability to circulate both in terrestrial cycles and in seabirds with their associated tick ixodes uriae [3234 ]. a study by comstedt. from 2011 determined that b. garinii was found in norwegian populations of seabirds and i. uriae. sequencing of the intergenic spacer (igs) from b. garinii strains has indicated that the seabird - associated b. garinii strains are more diverse than the terrestrial b. garinii strains. all strains included in the present study are collected from a geographic location with a high number of observed bird species. in mre and romsdal county, 290 bird species these birds represent a variety of different reservoir hosts for b. garinii, and the nesting on the island provides a possibility of transferring b. garinii into fauna, inducing circulation between migrating and stationary hosts. the high number of migrating birds along the coastal area of northwest norway enables introduction of b. garinii strains from multiple different geographic origins and multiple reservoir hosts and the process of circulation between reservoir hosts has been suggested to play a role in the genetic evolution of borrelia genospecies. some b. garinii strains from the present study are dlvs and tlvs of the strains from central europe, while others have levels 4 and 5 connections to the central european sts. within the norwegian b. garinii strains, some are slvs and dlvs, and some are more distantly related. this may indicate that the different strains have been introduced into the fauna by migrating birds at different times and that some strains might have been maintained by stationary hosts for some time. calculation of the nucleotide diversity (value) reveals that the values were at least two times greater for b. garinii than for b. afzelii. assuming a similar generation time between b. garinii and b. afzelii, the different reservoir hosts might have influenced the evolutionary rate differently between genospecies. the limited geographic dispersal of b. afzelii strains has allowed them to evolve differently between geographic locations, but the nucleotide diversity within a geographic location is relatively low. b. garinii associated with migratory birds is subjected to a higher degree of ecological determinants, and the nucleotide diversity may reflect that strains have been introduced to the fauna at different times. the fourth deep branched cluster contains one b. burgdorferi s.s. isolate and its associated reference species the low prevalence of this genospecies, in combination with certain challenges associated with amplification across all eight loci, caused this strain to be the only b. burgdorferi s.s. the number of polymorphic sites (ps) and the nucleotide diversity () were calculated to further describe the borrelia strains. these point mutations have a minor contribution to the genetic diversity between concatenated dna sequences from northwest norway compared to other worldwide strains. most of the point mutations are silent or missense mutations with little or no effect on the genetic function. the ratio between nonsynonymous substitutions and synonymous substitutions (dn / ds) < 1 indicates that the number of synonymous substitutions is higher than the number of nonsynonymous substitutions. this indicates that the genes are not under selective pressure and are suitable for mlsa analysis. the index of association (ia) describes the influence of recombination in a population and was calculated in two ways : using the classical method defined by smith. and using the standard method defined by haubold and hudson [38, 39 ] the calculated values for this population were iaclassic = 2.2054 and iastandard = 0.3151, and significant linkage disequilibrium was detected. estimation of the index of association indicated that the population is clonal and that recombination has not played an important role in the evolution of this population. multilocus sequencing analysis has provided knowledge about the genetic variations between borrelia genospecies originating from i. ricinus ticks from northwest norway. this is the first multilocus sequence analysis of borrelia strains identified from norwegian i. ricinus ticks, and the 50 borrelia strains from norway represent a large contribution to the mlst database. this enabled comparative studies between norwegian and european strains, thus creating a better understanding of the strain development of borrelia genospecies in different geographic locations in europe. the high level of divergence in the b. afzelii population was expected, but the divergence within the b. garinii population differs from previous mlst / mlsa studies. migrating birds are constantly introducing new strains to the fauna along the coastline in northwest norway, and circulation between migrating and stationary hosts would influence the strain diversity. some of the b. garinii strains show signs of being newly introduced to the fauna while others have most likely been maintained in stationary hosts for a longer period of time.	characterisation of borrelia strains from ixodes ricinus ticks is important in the epidemiological surveillance of vector - borne pathogens. multilocus sequences analysis (mlsa) is a molecular genotyping tool with high discriminatory power that has been applied in evolutionary studies and for the characterisation of borrelia genospecies. mlsa was used to study genetic variations in borrelia strains isolated from i. ricinus ticks collected from the woodlands in skodje. the results demonstrate that the 50 borrelia strains were separated into 36 sequence types (sts) that were not previously represented in the mlst database. a distance matrix neighbour - joining tree (bootstrapped 500 iterations) showed four deeply branched clusters, and each deeply branched cluster represented one borrelia genospecies. the mean pairwise genetic differences confirm the genospecies clustering. the combination of alleles separates the borrelia strains from northwest norway from the strains in the mlst database, thus identifying new sts. although a highly divergent b. afzelii population could be expected, the heterogeneity among the b. garinii strains is more unusual. the present study indicates that the circulation of strains between migrating birds and stationary birds in this coastal region may play a role in the evolution of b. garinii strains.
several catastrophic epidemics resulting from the consumption of food contaminated by mehg have highlighted the potentially disastrous effects of mehg on living organisms. mehg is a potent neurotoxicant which affects both the developing and mature cns [5, 6 ]. in infants, mehg causes widespread and diffuse damage, whereas focal damage is caused in the adult brain. in adults, chronic mehg poisoning results in the degeneration of the sensory cerebral cortex and the cerebellum, thereby leading to severe neurological disturbances, such as cerebellar ataxia and paresthesia, sensory and speech impairment, and the constriction of the visual field [4, 7, 8 ]. the pathological changes involve general neuronal degeneration with gliosis in the calcarine, and precentral and postcentral areas of the cerebral cortex, as well as the loss of granular cells in the cerebellar cortex. in biological systems, mehg exists only at a very low concentration as a free, unbound cation which can bind to sulfhydryl groups (-sh) of amino acids with a very high affinity (log k in the order of 1523). this affinity of hg for sulphur and -sh groups is a major factor underlying the biochemical properties of mehg, which, consequently, leads to its interference with the enzyme activities of several cellular targets. in the marine ecosphere, mehg is sustained [11, 12 ] and, after bioaccumulation, is introduced into the human population through the dietary intake of fish and seafood products. mehg toxicity due to the consumption of adulterated fish represents a major public health issue. greater fish consumption in many cases is paralleled by increased mehg intake ; however, conversely, lower maternal seafood intake has also been associated with higher risk for a suboptimal developmental outcome. according to the avon longitudinal study of parents and children (alspac), the authors reported that maternal seafood intake during pregnancy of less than 340 g per week was associated with an increased likelihood for their children to fall into the lowest quartile for verbal intelligence quotient (iq) when compared with mothers who consumed more than 340 g of seafood per week. though hg consumption was not assessed in this study, it is reasonable to assume that greater fish consumption was paralleled by increased mehg intake. moreover, several discrepancies in health outcomes in fish - eating populations have been reported, such as neurodevelopmental impairments in new zealand [1820 ] and the faroe islands [21, 22 ], as opposed to the beneficial effects noted in canada, the seychelles [24, 25 ], peru, and the united states [17, 2730 ]. additionally, laboratory studies have also shown that dietary factors, such as selenium, cysteine, protein, fat, fiber, and vitamin contents can modulate the toxicity and excretion of mercury [31, 32 ]. a previous study has also shown a significantly higher rate of fecal excretion as well as a lower degree of mehg accumulation in the brains of rats fed naturally contaminated fish as compared to rats fed fish containing chemically added mehg. the above - mentioned studies indicate that, in addition to intrinsic, genetic factors, the phenotypic responses to mehg exposure may ultimately depend on a number of complex interactions within biological systems involving both mercury and various dietary factors. it is therefore important to study the effect(s) of confounding dietary factors that occur when fish is consumed on mehg distribution and neurotoxicity. in this respect, it must be noted that different types of fish accumulate different concentrations of nutrients and contaminants [3436 ]. therefore it is of considerable interest to determine how each component acts individually (as well as with others) and influences the potential risk from mehg exposure. these cellular and molecular mechanisms of mehg action, as well as the underlying processes of its interaction with dietary components have yet to be defined, especially in specific central nervous system (cns) targets. accordingly, this paper focuses on studies directed toward estimating the effect(s) of dietary modifiers on mehg neurotoxicity, potentially providing information about critical cellular mechanisms responsible for conferring neuroprotection from a diet that includes mehg - contaminated fish. the disruption of redox cellular homeostasis by an excess of ros formation, which leads to cumulative oxidative stress appears to play a key role in the in vivo pathological process of mehg intoxication [3742 ]. conversely, several studies have demonstrated the partial amelioration of mehg toxicity in the presence of antioxidants by the inhibition of ros generation [40, 43, 44 ]. although the critical role of oxidative stress in the pathogenesis of mehg cytotoxicity has been clarified, the molecular mechanisms underlying mehg - mediated oxidative stress have not yet been fully elucidated. a major source of mehg - induced increases in ros generation may be the mitochondrial electron transport chain. the damaged mitochondrion increases oxidative stress, leading to a decrease in defense mechanisms, such as reduced gsh content, which represents one of the principal endogenous antioxidants. in addition, binding to gsh is reported to be responsible for the excretion of mehg. therefore, decreased gsh levels usually parallel increased oxidative stress due to mehg exposure [4549 ]. however, two epidemiological studies associating oxidative stress and mehg exposure [50, 51 ] have shown both an increase and a decrease in gsh levels with increased total hg levels. this suggests that mehg can increase ros which may either inhibit gsh levels or initiate an adaptive response to oxidative stress by increasing gsh levels. moreover, studies of human populations, although of direct interest, can not be controlled for multiple confounding variables. this obstacle can be overcome by conducting studies on laboratory animals ; such investigations can identify the mechanisms of action by which neurotoxicants and neuroprotectants interact. upregulation, or the induction of an increased synthesis of gsh, has been reported to provide neuroprotection against mehg - induced neurotoxicity. a similar alleviation in mehg - induced cytotoxicity and oxidative stress has been reported with n - acetyl cysteine (nac) supplementation [39, 5355 ]. the mechanisms involved in protection afforded by nac include increased intracellular gsh [54, 55 ] as well as a transient increase in the urinary excretion of mehg, which was shown to cause a decrease in the level of mehg in both the adult brain and the fetus [53, 56 ]. in addition, the increased amount of gsh in cortical, as compared to cerebellar, astrocytes has been reported to account for the increased mehg - induced ros production in cerebellar astrocytes. conversely, the depletion of intracellular gsh with diethyl maleate (dem) has been reported to increase cell - associated mehg and mehg - induced ros [48, 54, 55 ]. the underlying mechanism of this process involves the conjugation of free sulfhydryl groups of gsh with dem, which results in the distinct depletion of gsh. also, gestational exposure to mehg has been reported to cause the dose - dependent inhibition of cerebral gsh levels, an outcome which could be correlated with increased lipid peroxidation in the pup brain. these biochemical alterations were found to endure even after hg tissue levels decreased, thus indicating permanent functional deficits observed after prenatal mehg exposure as well as an additional molecular mechanism by which mehg induces prooxidative damage in the developing cns. in summary, changes in intracellular mehg content with gsh modulation provide an explanation for the increased susceptibility of certain cell types towards mehg - induced oxidative stress [54, 55 ]. dha cis-4,7,10,13,16,19-docosahexaenoic acid, is one of the most abundant polyunsaturated fatty acids (pufa) in the phospholipid fractions of the mammalian brain [58, 59 ]. both seafood and breast milk serve as major dietary routes of mehg [60, 61 ] and dha [6265 ]. the ability of dha to affect ros is controversial, as several contrasting studies have documented the ability of dha to decrease the level of lipid peroxide [6668 ] and to cause free - radical - mediated peroxidation in the brain [6971 ]. these studies have demonstrated the beneficial effects of dha on using a dha - enriched diet against mehg - induced decreases in serum albumin levels, changes in mitochondrial membrane potential, and developmental defects. however, other contradictory studies have reported no protection against mehg - induced behavioral defects [75, 76 ]. it is therefore important to identify the biochemical mechanisms involved in the dha protection against mehg neurotoxicity. kaur and colleagues [77, 78 ] demonstrated that pretreatment with dha was associated with reduced cell - associated mehg in neuronal cell lines and primary cells. in addition, decreased ros and unchanged gsh levels were found in primary cultures, whereas increased ros and gsh depletion were found in c6 cells [77, 78 ]. these differences with respect to the effect of dha on oxidative stress could be due to the fact that the growth of cancerous cells is inhibited by dha as compared to noncancerous cell types [71, 79 ]. indeed, another recent study has shown that fish oil offers significant dna protection as well as anti - inflammatory effects in the absence of changes in gsh levels. these observations strongly suggest that dha may neuroprotect against mehg - induced ros generation even in the absence of significant changes in gsh levels. selenium (se) is an essential trace element known to accumulate in significant amounts in numerous species of seafood [80, 81 ]. the majority of se in fish is in the organic form, selenomethionine (sem) [82, 83 ], and is more bioavailable than are inorganic forms. the modulating effect of se on mehg toxicity was discovered when researchers observed that marine mammals could accumulate exceptionally high concentrations of hg and se compounds without displaying obvious symptoms of intoxication [86, 87 ]. several subsequent studies later confirmed that the toxic effects of both organic and inorganic hg were prevented by se compounds [8892 ]. treatment with different se compounds has been shown to effectively protect cells against different toxic effects induced by mehg exposure, such as cytotoxicity, fetotoxicity, neurotoxicity, and developmental and neurobehavioral toxicity [9397 ]. in addition, se deficiency has been shown to potentiate the adverse effects of mehg toxicity in rodents [98, 99 ]. with regard to epidemiological studies and se content, it is important to note that faroe islanders, by virtue of a whale meat diet, are generally exposed to mehg levels that are in excess of se levels, whereas the seychellois are largely ocean fish consumers, and se molar concentrations tend to greatly exceed mehg concentrations in this seafood source. in addition, the dietary se status in the new zealand population was extremely poor at the time of the study. this distinction could be one explanation for the different effects noted in these studies, although additional evidence is needed to support this hypothesis. therefore, developing a better understanding of the mechanisms associated with the interaction of mehg and se is of particular necessity. several studies have indicated that the mechanism underlying se 's ability to ameliorate mehg toxicity is related to an antioxidant effect [104108 ], which includes the formation of gsh, higher glutathione peroxidase (gpx) activity, increased selenoprotein levels [110112 ], and the reduction of organic hydroperoxides [113115 ]. additionally, studies have shown that binding of mehg [116, 117 ] and the formation of a highly stable organic mehg - selenocysteine complex also influence the accumulation of mehg in tissues [118121 ] and the uptake of mehg in cells [114, 122124 ]. furthermore, se is known to enhance the excretion of mehg [56, 125 ], and a recent study has shown that sem can demethylate mehg under physiologically and environmentally relevant conditions. hence, the interactive effects between mehg and sem result in reduced cell - associated mehg and prooxidant response from mehg. seafood serves as a source of vitamins, with estimates ranging between 4.84 and 17.90 g vitamin e per gm of fish, which makes this vitamin the most significant physiologic membrane - associated antioxidant available from seafood. trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water soluble analog of vitamin e, serves as a better antioxidant than vitamin e [129, 130 ] due to its improved access to the hydrophilic compartments of the cells, as well as its stoichiometric properties. trolox scavenges free radicals [59, 133137 ] via the h - donating groups [128, 138 ]. treatment with trolox has been reported to protect against mehg - induced cytotoxicity, the decrease in mitochondrial electron transport system enzyme activities, and the increase of mrnas of antioxidant enzymes [108, 140 ]. trolox treatment has also been shown to reverse ros induction by mehg in primary astrocyte cultures and to prevent mehg - induced oxidative stress, where the modulating effect of trolox on cellular ros levels was not accompanied by changes in cellular mehg, gsh, or mtt activity. these findings indicate that trolox affords protection against ros by the direct quenching of free radicals and not by mehg chelation or by the induction of increased levels of gsh or mitochondrial enzymes. in fact, it has been previously shown that in vivo protection with trolox does not affect intracellular gsh [142, 143 ] or mehg levels. the recognition of the protective effects of trolox and the identification of its mechanisms via in vitro models establish that vitamin - dependent antioxidant defences are important factors in specific cells for attenuating the neurotoxic effects of a mehg - contaminated fish diet. fish is not only an excellent nutritional source of protein, vitamins, zinc, and other minerals, but it is also a source of exposure to mehg [144, 145 ]. one of the leading controversies in the mehg literature originates from advisories concerning the consumption of fish and from uncertainties in documentation from various regulatory agencies regarding the effects of mehg. the joint fao / who expert committee on food additives reported in 1978 that the fetus may be more susceptible to mehg toxicity than the adult. the united states white house in 1998 convened an international workshop where a variety of possible uncertainties and confounders important to mehg toxicity evaluation were discussed. their conclusions stated, even when dietary stresses and co - exposures to other chemicals could plausibly enhance or alter risk, it was still deemed that there are inadequate data on this subject to draw meaningful conclusions at this time. later, in 2000, the national academy of sciences committee reported that, 60,000 children in the united states were at risk as a result of prenatal exposure. however, no justification or explanation for that conclusion was provided [16, 150 ]. the issue that poses a significant dilemma for both consumers and regulatory authorities is whether fish consumption should be encouraged for its nutritional benefits to the developing brain or, conversely, whether fish consumption should be discouraged due to the possible adverse effects of mehg on the developing cns. this nutrition versus neurotoxicity controversy can be addressed by estimating the effects of dietary factors on mehg - induced toxicity as well as by determining the mechanisms behind such effects. a thorough assessment of coexposure from dietary nutrients as well as neurotoxic exposures would offer valuable information for accurately determining the risks and benefits of fish consumption [151, 152 ]. this paper explores the mechanisms associated with mehg and dietary nutrients obtained from the consumption of seafood. the toxicity of mehg has been reported to be caused by a reduction in the amount of intracellular gsh [45, 46, 48 ], which leads to the augmentation of ros formation [37, 4044, 153 ]. this paper investigates the effects of mehg on oxidative stress and details the role played by gsh in modifying these effects. it also identifies the biochemical mechanisms underlying exposure to gsh, dha, se, trolox, and mehg, where these modifiers have been shown to effectively decrease mehg - induced ros (figure 1). in addition, it is important to note that the interaction between these dietary nutrients may have an effect on overall toxicity. for example, the benefits from se against mehg toxicity can be influenced by the intake of long - chain, polyunsaturated fatty acids (lcpufas) [65, 154 ]. it has also been shown that the shape of the dose - effect curve for hg is dependent upon the co - exposure of dietary components such as se and vitamin e. this paper, concludes that gsh, dha, se and trolox are strong confounders in the association of mehg toxicity and that the interaction between them may affect the cellular oxidative status. thus, it is necessary to consider different confounders and the various mechanisms by which they interact with hg when investigating the potential beneficial effects of fish consumption. indeed, doing so would provide valuable insight for developing a better understanding of the benefits and risks of fish consumption, acknowledging both the proven beneficial nutrients as well as the potentially dangerous contaminants contained in this important food source. furthermore, such information would also assist public health authorities as they seek to advise the populace and as they undertake efforts to formulate appropriate dietary recommendations for consumers of fish and seafood products.	methylmercury (mehg), an environmental toxicant primarily found in fish and seafood, poses a dilemma to both consumers and regulatory authorities, given the nutritional benefits of fish consumption versus the possible adverse neurological damage. several studies have shown that mehg toxicity is influenced by a number of biochemical factors, such as glutathione (gsh), fatty acids, vitamins, and essential elements, but the cellular mechanisms underlying these complex interactions have not yet been fully elucidated. the objective of this paper is to outline the cellular response to dietary nutrients, as well as to describe the neurotoxic exposures to mehg. in order to determine the cellular mechanism(s) of toxicity, the effect of pretreatment with biochemical factors (e.g., n - acetyl cysteine, (nac) ; diethyl maleate, (dem) ; docosahexaenoic acid, (dha) ; selenomethionine, sem ; trolox) and mehg treatment on intercellular antioxidant status, mehg content, and other endpoints was evaluated. this paper emphasizes that the protection against oxidative stress offered by these biochemical factors is among one of the major mechanisms responsible for conferring neuroprotection. it is therefore critical to ascertain the cellular mechanisms associated with various dietary nutrients as well as to determine the potential effects of neurotoxic exposures for accurately assessing the risks and benefits associated with fish consumption.
complexity in chemistry ; reaction networks, coupled equilibria, spatiotemporal compartmentalization, or feedback loops often result in emergent behavior characterized by responsiveness, adaptivity and nonlinearity ; life being the prime example. even though our creations can not yet rival those of nature, the rise of interest in systems chemistry gives hope that the gap will decrease as we gather understanding and devise new mechanisms to create and control complexity. one of the more successful methods to generate complex (supra)molecular systems is dynamic combinatorial chemistry (dcc). in this approach, a few small building blocks react reversibly with each other, giving rise to mixtures of much more complex library members, together constituting a dynamic combinatorial library (dcl). in its relatively short history, it has led to practical outcomes, such as discoveries of biologically active compounds or responsive materials, as well as to discoveries of fundamental value, such as emergence of self - replicating molecules or complex reaction networks and cascades. addition of a second type of reversible chemistry not only adds another layer of complexity, but also provides an additional handle to control it. however, only a small fraction of reported work takes advantage of this strategy, and only a handful describe three or more dynamic covalent chemistries in a single system. this situation stands in a stark contrast with supramolecular systems, where several different interaction motifs are often used simultaneously. combined combinatorial chemistries can be orthogonal (scheme 1a), when one functional group can only be involved in formation of one covalent bond type, or promiscuous (scheme 1b), which means that some of the functionalities can form more than one type of dynamic covalent bonds. disulfide and hydrazone exchange are a pair of orthogonal chemistries, as in aqueous solution thiols do not form stable adducts either with aldehydes, or with hydrazides. in such cases the two chemistries operate completely independently, unless they are coupled by an independent interaction, e.g., noncovalent bonds. on the other hand, libraries based on thiol disulfide exchange can easily communicate with thioester - based libraries, as both reactions involve promiscuous thiol building blocks. depending on exact chemistries used, reaction conditions may be tuned in such a way that exclusively one type of exchange is active, or that two or more chemistries operate simultaneously (scheme 1a). this is however more often defined by the nature of the exchange chemistries, than by the intentions of the experimenters. in the case of disulfides and thioesters, for example, the two chemistries tend to only work simultaneously at mildly basic ph. in contrast, hydrazone exchange, which normally operates at moderately to strongly acidic ph, was only active simultaneously with disulfide exchange at the cost of both reactions being slow. different exchange types are represented by bond and building block (bb) shapes, whereas colors denote bb identity. for clarity (a) orthogonal chemistries : under conditions (1) only one type of exchange is active, under conditions (2) only the other, whereas under conditions (3) both exchanges operate simultaneously. under all conditions bbs exchange only within the same type of chemistry. however, the number of components involved in each exchange pool remains constant. (c) antiparallel chemistries (this work) : bbs participate in both chemistries, but the ratio of the two chemistries can be tuned by altering the system conditions. combining exchange chemistries that can communicate leads to another interesting possibility : if the two exchange pools share a building block, increase of its amount in one pool necessarily depletes it in the other one. in other words, the distribution of covalent bond types is reflected by the composition of the dcl, leading to the concept of antiparallel chemistries (scheme 1c). the term antiparallel reflects that both reactions can take place at the same time (thus parallel) but occur at each other s expense (hence anti). in a system that comprises two parts which share a constituent that can be shifted from one to another by an external parameter, a new level of control emerges. together with the thermodynamic control inherent to the dcl itself, its composition now also depends on the external parameter, which is in the hands of the experimenter. thus, in antiparallel chemistries the two reactions can occur simultaneously and communicate through a common reactant, which is distinct from the situation in orthogonal chemistries in which all reactions operate independently of each other. in our design of antiparallel chemistries we decided to combine thiol disulfide and thio - michael exchange (figure 1). the choice stems from the fact that both chemistries involve thiols, but the library members themselves require sulfur atoms to be in different oxidation states. disulfides form from thiols by oxidation, whereas formation of thio - michael adducts does not result in oxidation of thiols. therefore, the oxidation state of the library controls the disulfide / thio - michael ratio. in a fully reduced library there can be only thio - michael adducts, while oxidation increases the amount of disulfides at the expense of the thio - michael adducts until the library is fully oxidized, and the thio - michael adducts are replaced by disulfides. such antiparallelism of these two chemistries is possible only because the thio - michael reaction has different number of thiols on both sides of the equilibrium, allowing for depletion of its reaction pool by thiol removal. such operation would not be possible with e.g., thioester exchange, where both sides of the equilibrium contain the same number of each species. for the thiol building block we chose dithiol a, already known to form a series of macrocycles upon oxidation, while instead of a classical michael acceptor we decided to use bc, previously reported by joshi and anslyn (figure 2a). the latter, being a michael acceptor with one thiol group already present (and unable to dissociate into thiol and alkyne), is in fact bivalent, which means that in combination with a it can give rise to a mixture of linear and macrocyclic compounds, thus being a promising starting material for making diverse dcls. upon mixing the starting materials in the absence of oxidants we expected a mixture of linear and macrocyclic thio - michael mono- and bis - adducts would form. mixing fully oxidized a with bc, on the other hand, should not lead to any changes, as disulfides do not form adducts with michael acceptors, while at intermediate oxidation levels the system should contain the thio - michael adducts, disulfides and possibly a number of species containing both types of bonds. (note that in fully oxidized libraries, building block c has to be present as a single michael adduct as it can not undergo a -elimination. thus, such dcls will contain 2/3 of their a, b content in the form of disulfides and 1/3 as single michael adducts. note that for michael acceptors that can undergo complete -elimination, fully oxidized dcls will not contain any michael adducts or free thiols.) (a) building blocks (above) and characteristic representatives of thio - michael adducts (left), disulfides (right), and intermediate species (middle) ; (b) chromatograms of the antiparallel dcls at different oxidation levels : fully reduced (bottom), 50% oxidized (middle), and fully oxidized (top) ; (c) heat map plot showing the abundances of the library constituents depending on the oxidation level (shade represents the peak area normalized to the maximum amount the particular species reach ; the numbers next to the species show the red / ox ratio of the sulfur atoms in their structures). to test our hypotheses, we performed two series of experiments, the first to see the outcome of mixing of bc with a at various levels of oxidation, and the second to see whether the system can be reversibly reduced and oxidized. in the first series we investigated libraries initially containing equimolar amounts of a and bc (both 2.5 mm), at oxidation levels ranging from we prepared these libraries by mixing 5.0 mm solutions of a and fully oxidized a (an) to obtain the desired redox level, followed by the addition of an equal amount of 5.0 mm bc (all components were dissolved in an aqueous borate buffer, ph = 8.2). after mixing, until equilibrated (kinetic experiments showed no changes after 24 h, except for the fully oxidized library), and subsequently analyzed by uplc, while the library members were identified by uplc - ms. control experiments revealed that the uv response is a linear function of the concentrations of the various library members (see supporting information (si) section 3). the results show that upon mixing a and bc a diverse library is formed rapidly, containing 20 different detectable species. the expected linear or macrocyclic thio - michael adducts accounted for a large part of the library (the dominant species are b and ac, as visible in figure 2b, bottom). in the fully oxidized library (figure 2b, top), also expectedly, the disulfides stemming from a and the initial michael acceptor bc dominate, while the presence of exchange products (b2, cac, and bab) can be explained by traces of thiols remaining after oxidation of a. due to low exchange rates, the data for the fully oxidized library may differ from what would be present at equilibrium, but, as later analysis will show, the difference is small in the worst case and the general trends hold at all oxidation levels. solutions at the intermediate oxidation levels (e.g., 50%, as shown in figure 2b, middle), together with the thio - michael adducts and the disulfides, also contain a number of species which contain both kinds of covalent bonds, altogether forming libraries of over 30 different compounds. plotting the normalized peak areas of the library constituents against the oxidation level (figure 2c) shows the thiols and the michael adducts generally reach their maximum concentrations when the system is fully reduced, and gradually diminish as the oxidation level increases. disulfides and michael acceptors (cac and bc) follow exactly the opposite pattern, and reach their maximum concentrations at high oxidation levels. the intermediate species, which contain both thio - michael and disulfide linkage, are nearly absent at the two extremes. altogether, these observations confirm the initial hypothesis that, in a system comprising thiols and michael acceptors, the distribution of the bond types and therefore the composition of the library depends on the oxidation level. in the second series of experiments we tested the redox reversibility of the system ; i.e., whether the library composition can be controlled by an external input, in this case reducing or oxidizing agents. for that purpose we prepared fully oxidized and fully reduced libraries of a and bc, in a similar way as described for the first series. after 48 h equilibration, which led to the same compositions as described previously, samples of the fully reduced library were oxidized by nabo3 or i2, and samples of the fully oxidized library were reduced by tcep or dtt. for each reagent, 0.3, 0.5, 0.7, or 1.0 equiv the libraries were again left to equilibrate for 48 h and then analyzed by uplc. the results (figure 3) show that the system is indeed redox reversible and also that the reactions proceed without any side products, as no new peaks appeared in the chromatograms. therefore, the library can be switched to any intermediate oxidation level and the corresponding composition by simply adding redox agents, allowing for easy external control. only the fully oxidized library does not equilibrate readily because the disulfide exchange is catalyzed by thiolate anions, which are absent under these conditions. (a) comparison of 50% oxidized libraries obtained in five different ways (from top to bottom) : addition of reducing agents (tcep or dtt) to 100% oxidized dcl ; mixing half - oxidized a with bc ; addition of oxidizing agents (i3 or nabo3) to a 0% oxidized dcl. (b) heat map showing the distribution of the library constituents in the 50% oxidized libraries. our attempts to rationalize the behavior of the system revealed an interesting phenomenon : as shown above, an macrocycles and bc dominate at 100% oxidation, whereas b and ac adducts are main species at low oxidation levels (figure 4). however, we can imagine the opposite scenario, where b2 and cac would be the main species in a fully oxidized dcl, with a and bcb dominating the unoxidized library. their effect becomes clear as we analyze the equilibria for oxidized (eq 1) and reduced (eq 2) dcls, connecting the two alternative scenarios:12 three - dimensional plot showing abundances of representative dcl member families coupled by antagonistic relations, as a function of the oxidation level of the library. the diameter of the circles represents the summed peak area of library members connected by a vertical edge. as we can see, the left sides of both equilibra have 2n molecules, whereas there are 2n + 1 molecules on the right sides. thus, it is entropically preferred to shift the equilibrium to the right side, i.e., in favor of, respectively, an and bc, and b and (ac)n. interestingly, the composition of the system does not follow monotonically from one oxidation extreme to the other. for example, b2 and cac reach their maximum concentrations at partial reduction while they are both fully oxidized species (figure 2c). to better understand this counterintuitive behavior, we found it informative to plot different library member families onto a single three - dimensional graph, represented as a cube (figure 4). one axis of the plot corresponds to a : b ratio within a library member, another to the c content. the third axis corresponds to the oxidation level : as it increases, the thiol : disulfide ratio decreases and double thio - michael adducts become single adducts. species sharing an edge of the cube are antagonists, as they compete for the same building blocks or oxidation state. the latter results from the wiring of the network, which makes the antagonistic effects distinct from previous reports based solely on the competition between library members for common building blocks involving only one type of chemistry. library members can be mapped onto the cube as a function of their composition and oxidation state of their sulfur atoms. plotting the sum of all library members corresponding to different oxidation levels for the four composition extremes as a function of library oxidation reveals that the equilibria 1 and 2 dominate only at the extreme oxidation levels. in fact, the diagonals connecting the agonistic species at the favored sides of these equilibria are perpendicular to each other. therefore, compounds like b2 that, at full oxidation, suffer from antagonism by entropically favored compounds (equilibrium 1) start to benefit from agonism by compounds that become entropically favored at lower oxidation levels. hence, despite being fully oxidized themselves they benefit from partial reduction of the mixture. thus, the concentration of library members is a complex function of the structure of the building blocks, the wiring of the molecular network, and the experimental conditions. while the design of the thio - michael system shown in figure 2 is somewhat unconventional, we also performed similar experiments on a classical michael acceptor d ((e)-4-phenylbut-3-en-2-one), while retaining the dithiol a. the results (si, pages s39s50) show that the concept also applies to more traditional thio - michael additions, in which only a single thiol adds to the michael acceptor. to conclude, we have designed a system in which two chemistries, namely thiol disulfide exchange and thio - michael exchange, operate simultaneously, giving rise to diverse dcls. as both chemistries use the same building blocks, as one exchange pool grows, furthermore, as the two pools require sulfur atoms at different oxidation level, external control of their ratio is possible using reducing and oxidizing agents. we envisage that dynamic covalent antiparallelism should be applicable to other pairs of dynamic covalent chemistries, e.g., disulfide / thiazolidine, or the recently developed dithioacetal / disulfide system. especially exciting should be a combination of antiparallel, orthogonal, and communicating chemistries, allowing for complex and addressable feedback between different subsystems. the particular system studied has also shown how the antiparallelism of the two chemistries, combined with opposing entropic effects, gives rise to a complex network of interactions, resulting in nonlinear changes in the library composition in response to the external stimulus. externally addressable complexity achieved in such way should prove useful in functional screening of dcls, where the library can be biased toward a desired connectivity type, rather than just building block composition. from the systems chemistry perspective, we are excited to see how antiparallelism creates molecular systems that can adapt to environmental changes by switching to the type of chemistry better fitted for the new conditions. this emergent behavior to some extent resembles homeostatic processes in living organisms, or switching between aerobic and anaerobic metabolisms. water was doubly distilled prior to use. 4-mercaptobenzoic acid (technical grade, 90%) and 3-butyn-2-one (96%) used for the synthesis of bc were purchased from sigma - aldrich and acros organics, respectively, and used without further purification. boric acid and potassium hydroxide utilized for the preparation of buffers and ph adjustment were obtained from acros organics and merck chemicals, respectively. sodium perborate, potassium iodide, dithiothreitol (dtt), and tris(2-carboxyethyl)phosphine (tcep) used for the reduction / oxidation of a and libraries were purchased from sigma - aldrich. acetonitrile (ulc / ms grade) and water (ulc / ms grade) were obtained from biosolve bv. the 50 mm borate buffer was prepared from boric acid dissolved in doubly distilled water, and adjusted with 1.0 m koh to ph 8.2. afterward, it was degassed by nitrogen purging under reduced pressure for 60 min. libraries were prepared in clear hplc glass vials (12 32 mm) closed with teflon - lined snap caps purchased from jaytee. library solutions were stirred using teflon - coated microstirrer bars on a magnetic stirrer at 1100 rpm. we prepared a 5.0 mm stock solution of bc, a 10 mm stock solution of a, and a 10 mm stock solution of nabo3. equal volumes of a and nabo3 solutions were mixed to obtain 5.0 mm oxidized a, and left stirring for 3 h before further use. simultaneously, a was diluted twice with buffer solution to obtain 5.0 mm unoxidized a. libraries were prepared by mixing adequate volumes of bc, reduced a, and oxidized a (as listed in table s1). for uplc and uplc - ms analyses, 3 l samples were drawn from solutions and diluted with 6 l of dmso prior to injection. a 6.11 mm stock solution of bc was prepared ; 5.0 mm stock solutions of reduced and oxidized a were used from previous experiments. solutions were prepared by mixing 550 l of either reduced or oxidized a and 450 l of bc to give equimolar mixtures with a final concentration of 2.75 mm (of each building block). solutions of redox agents, dithiothreitol (dtt), tris(2-carboxyethyl)phosphine (tcep), sodium perborate (nabo3), and iodine in potassium iodide (ki + i2) (25 mm each), were prepared right before use. they were mixed with the above solutions (reduced or oxidized) of a and bc in proper ratios (as listed in tables s5 and s6) to obtain dcls with oxidation level set as 0%, 30%, 50%, 70%, or 100%, and left for 2 days to equilibrate.	the ability to design reaction networks with high, but addressable complexity is a necessary prerequisite to make advanced functional chemical systems. dynamic combinatorial chemistry has proven to be a useful tool in achieving complexity, however with some limitations in controlling it. herein we introduce the concept of antiparallel chemistries, in which the same functional group can be channeled into one of two reversible chemistries depending on a controllable parameter. such systems allow both for achieving complexity, by combinatorial chemistry, and addressing it, by switching from one chemistry to another by controlling an external parameter. in our design the two antiparallel chemistries are thiol disulfide exchange and thio - michael addition, sharing the thiol as the common building block. by means of oxidation and reduction the system can be reversibly switched from predominantly thio - michael chemistry to predominantly disulfide chemistry, as well as to any intermediate state. both chemistries operate in water, at room temperature, and at mildly basic ph, which makes them a suitable platform for further development of systems chemistry.
semm first introduced the laparoscopic method for appendicectomy in the early 1980s. since then laparoscopic appendicectomy (la) was made popular by various surgeons and preferred over the open method due to its inherent advantages. however, this technically demanding procedure requires increased surgical time and general anesthesia with positive pressure ventilation. also, this is an expensive procedure and requires an advanced surgical set - up and skilled surgeons, which prevent its use as routine practice in developing countries. the laparoscopic - assisted appendicectomy (laa) technique has all the advantages of the laparoscopic method at less expense than the completely laparoscopic technique, with a shorter operating time as an added advantage. the aim of our study is to evaluate the feasibility of laparoscopic - assisted appendicectomy (laa) using the two - port technique under local anesthesia in adults. as a pilot study we included a selected group of patients with low body mass index (bmi) and uncomplicated appendicitis. we conducted a prospective study over a two - month period where laa was performed on 12 patients (seven female and five male) of asa grade i or ii, who presented with acute appendicitis. inclusion criteria included pain in the right iliac fossa, with muscle guarding, tenderness at mcburney 's point, vomiting, fever, leukocytosis, and age more than 18 years. all the patients had a plain abdominal radiograph, routine blood tests, and a sonographic examination. patients with generalized peritonitis, appendicular abscess or perforation, and a palpable mass were excluded from the study. the patients who were found to have gangrenous or perforated appendicitis under laparoscopic view were excluded from this study and converted into the open technique. all the patients were explained about the procedure and the possible conversion into open technique and written consent was taken. in the operation theater (ot), an intravenous catheter was placed in the patients and ringer lactate infusion was started. ondansetron (100 g / kg.), diazepam (200 g / kg.), and pentazocine (500 g / kg) were used as premedication. all patients were monitored for blood pressure (non - invasive), heart rate, electrocardiogram, and oxygen saturation by pulse oximetry. the infra - umbilical area and the mcburney 's point area were infiltrated with 1% lignocaine with adrenalin, keeping the dose under 6 mg / kg of body weight, to avoid lignocaine toxicity. the pneumoperitoneum was created using carbon - di - oxide and the pressure was kept at 11 mmhg. a 0 telescope was introduced through the umbilical port for complete examination of the abdomen. a 10 mm trocar in the mcburney 's point area was introduced under vision, for holding the tip of the appendix. the pneumoperitoneum was deflated after the exteriorization of the appendix through the trocar placed at the mcburney 's point. the cut end was painted with betadine (aqueous solution of 10% povidone - iodine). the appendicular stump was then reposed within the abdomen. the patients were started oral feed immediately postoperatively and solid food on the next day. twelve patients underwent laa with a mean age of 22.5 years ranging from 18 to 30 years, with a mean bmi of 19.25 kg / m ranging from 16.18 to 24.15 kg / m. the appendix was in the normal position in nine patients and retro - cecal in three patients. two cases required a 5 mm extra port to free the appendix from the adhesion. one case was converted to open procedure due to the presence of adhesions of inflammatory origin. no patients required conversion to general anesthesia with positive pressure ventilation or spinal anesthesia, and no patient developed signs of toxicity due to lignocaine. two patients needed supplementation of injection ketamine 10 mg for analgesia. no postoperative complications, including wound infection, use of local anesthesia for surgical procedures has been steadily refined since its introduction by koller in 1884, and it is now widely used in several surgical procedures like cholecystectomy, thyroid surgeries, and appendicectomy. limited access for exploration, systemic toxicity due to excess amount of local anesthetics, limited duration of anesthesia, fear of failure are the reasons for avoidance of using local anesthesia for surgical procedures. simplicity in using is the most important attraction for local anesthesia, provided the surgical procedure is limited in exploration and duration, and does not disturb the internal milieu of the human body. it avoids the complications related to spinal, epidural or general anesthesia as well as the patients fear about the stated procedures. the feasibility of open appendicectomy under local anesthesia was already established by sharma. they found it to be cost - effective, and it also carried little morbidity and could be safely used for all age groups. we performed laa under the mac technique, where local anesthesia was combined with sedation and analgesia. local anesthesia was used for inserting the trocars, and sedation analgesia for creating pneumoperitoneum and excision of the appendix. mac required less anesthesia time and avoided the related complications and cost burden of spinal or general anesthesia. it requires less postoperative analgesia and is associated with faster recovery leading to shortened postoperative hospital stay when compared with the open technique. it is associated with lower postoperative complications including wound infection, and better cosmetic effect than the open technique. however, other studies where the laa technique was adopted, reported a varied number of wound infection rates. however, it was more expensive than the open technique. performing laa under local anesthesia with the sedation analgesia technique made it even cheaper. due to its short surgical time and also shortened time requirement for administration of anesthesia (mac), its overall ot engagement duration was less, which allowed more number of operations to be taken up within the stipulated time limit. one important advantage of the laparoscopic proceeding was the outstanding overview of the abdominal cavity with the possibility of more accurate diagnosis and proper decision - making. two - port laa is a safe and effective alternative for the management of uncomplicated appendicitis. however, the results can not be generalized to overweight patients. when compared with the three port technique the overall cost is less with two - port laa and it produces an even better cosmetic effect, due to the lesser number of punctures involved. the two port technique under local anesthesia is safe in uncomplicated appendicitis among low bmi patients and should now be rigorously evaluated in a randomized controlled trial, to investigate any potential advantage of this method over the conventional laa techniques.	aim : nowadays laparoscopic - assisted appendicectomy using the two - port technique is gaining popularity due to its certain benefits over the open version. general anesthesia with positive pressure ventilation is the preferred mode of anesthesia in this technique. we conducted a pilot study using the two - port technique in adult patients, with uncomplicated appendicitis under local anesthesia, to evaluate its feasibility.materials and methods : in this prospective study 12 consecutive patients of asa grade i and ii, with a mean age of 22.5 years, suffering from acute appendicitis, were included. all the patients received ondansetron, diazepam, and pentazocine as premedication. monitored anesthesia care was given. the site of trocar insertions were infiltrated with 1% lignocaine with adrenalin. the pneumoperitoneum was created using carbon - di - oxide. after exteriorization of the appendix using the trocar, appendicectomy was performed as in the open procedure.resultseleven out of twelve patients were successfully operated using this method without converting it into an open method. two cases required an extra port to free the appendix from the adhesion. there were no intra- or post - operative complications present.conclusion:two-port laparoscopic - assisted appendicectomy under local anesthesia is a safe and effective method for uncomplicated appendicitis in adults, and the procedure is suitable where limited set - up for anesthesia is present.
the mouse fasl locus and known restriction sites were used to construct the targeting vector and diagnose homologous recombination in embryonic stem (es) cells and gene - targeted (fasl, fasl) mice. targeting knock - in vectors were made with the loxp / pgkneo / loxp cassette cloned into the pac1 site. targeting constructs for the mutant fasl mice (supplementary fig. all experiments with mice were performed according to the guidelines of the animal ethics committees of our institutions. mice were killed at 6, 12 or 20 weeks for analysis and further cohorts were monitored daily for morbidity and killed when showing signs of illness. tissues were fixed for microscopic analysis in 80% histochoice (amresco)/20% ethanol or 10% buffered formalin and embedded in paraffin and conventional histopathology was performed on hematoxylin plus eosin stained sections. for detailed methods for immunohistochemical staining, immunofluorescent staining, confocal microscopy, cell preparation, flow cytometric analysis, elisa, aicd, chromatography, viral infection and target cell killing, t cell proliferation assays and western blotting refer to the online methods version. statistical analysis was performed using the student 's t - test, log rank (mantel - cox) test for survival curves or one - way analysis of variance using turkey 's comparison test to compare multiple groups where appropriate. the mouse fasl locus and known restriction sites were used to construct the targeting vector and diagnose homologous recombination in embryonic stem (es) cells and gene - targeted (fasl, fasl) mice. targeting knock - in vectors were made with the loxp / pgkneo / loxp cassette cloned into the pac1 site. targeting constructs for the mutant fasl mice (supplementary fig. all experiments with mice were performed according to the guidelines of the animal ethics committees of our institutions. mice were killed at 6, 12 or 20 weeks for analysis and further cohorts were monitored daily for morbidity and killed when showing signs of illness. tissues were fixed for microscopic analysis in 80% histochoice (amresco)/20% ethanol or 10% buffered formalin and embedded in paraffin and conventional histopathology was performed on hematoxylin plus eosin stained sections. for detailed methods for immunohistochemical staining, immunofluorescent staining, confocal microscopy, cell preparation, flow cytometric analysis, elisa, aicd, chromatography, viral infection and target cell killing, t cell proliferation assays and western blotting refer to the online methods version. statistical analysis was performed using the student 's t - test, log rank (mantel - cox) test for survival curves or one - way analysis of variance using turkey 's comparison test to compare multiple groups where appropriate.	fas ligand (fasl), an apoptosis - inducing member of the tnf cytokine family and its receptor, fas, are critical for shutdown of chronic immune responses1 - 3 and prevention of autoimmunity4,5. accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice6,7 and humans8,9. fasl function is regulated by deposition in the plasma membrane and metalloprotease - mediated shedding10,11. we generated gene - targeted mice that selectively lack either secreted fasl (sfasl) or membrane - bound fasl (mfasl) to resolve which of these forms is required for cell killing and to explore their hypothetical non - apoptotic activities. mice lacking sfasl (fasls/s) appeared normal and their t cells readily killed target cells, whereas t cells lacking mfasl (faslm/m) could not kill cells through fas activation. faslm/m mice developed lymphadenopathy and hyper - gammaglobulinaemia, similar to faslgld / gld mice, which express a mutant form of fasl that can not bind fas, but surprisingly, (on a c57bl/6 background) faslm/m mice succumbed to sle - like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and considerably later in faslgld / gld mice. these results demonstrate that mfasl is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer whereas excess sfasl appears to promote autoimmunity and tumorigenesis through non - apoptotic activities.
williams campbell syndrome, also known as bronchomalacia, is a rare disorder characterized by a deficiency of cartilage in subsegmental bronchi, leading to distal airway collapse and bronchiectasis. there have been few reports about patients affected by saccular bronchiectasis, paracicatricial emphysema, and diminished cartilage. this report presents a 57-year - old woman with progressive dyspnea, cough, sputum production, and fever. the clinical and laboratory examination revealed that the patient had a respiratory infection due to bronchiectasis caused by williams although a rare syndrome, when patients signs and symptoms include recurrent respiratory infections, bronchiectasis, productive cough, and dyspnea, williams campbell syndrome should be included in the differential diagnosis. williams campbell syndrome is a rare disorder characterized by a deficiency of cartilage in subsegmental bronchi, leading to distal airway collapse and bronchiectasis campbell syndrome results from the absence of cartilage rings beyond the first and second bronchial divisions with resultant bronchiectasis, which typically affects the fourth- to sixth - order bronchi. the symptoms and prognosis ultimately depend on the extent of cartilage maldevelopment of the bronchi. although the syndrome has been best described in children with recurrent pneumonia and broncho - obstructive symptoms such as coughing and wheezing, there have been recent descriptions in adults as well.1,2 a 57-year - old woman presented in the emergency department complaining of progressive dyspnea for the previous 5 days. on clinical examination she had productive cough with clear sputum and intermittent fever. she was a lifelong nonsmoker but with multiple hospitalizations due to respiratory infections and asthma - like episodes from the age of 8 years. during the past 15 years the patient was treated symptomatically with antibiotics, nebulizer bronchodilators, and long - term oxygen therapy (2 l / min/24 hours). her family past medical history included her 30-year - old sister s sudden death from unknown reasons and her mother s infection from tuberculosis. at the time of admission her axillary temperature was 38c and blood pressure 120/70 mmhg. the patient was in respiratory insufficiency with an arterial ph of 7.46, pco2 of 56 mmhg, po2 of 72 mmhg, and hco3 of 30 mmol / lung auscultation revealed widespread bilateral crepitating and crackles over the lower part of both lungs. laboratory data showed an elevated erythrocyte sedimentation rate of 100 mm / h and a white blood count of 9000/l without peripheral blood eosinophilia, and c - reactive protein was 9.3 mg / dl. her chest x - ray demonstrated increased lung volumes and revealed multiple ring shadows in lungs that were almost symmetrical in distribution and more apparent in the mid and lower zones. there was the sense of air - fluid levels in some of them corresponding to cystic bronchiectasis. also, there was thickening of the interstitial network. total serum immunoglobulin (ig)e concentration was elevated at 2500 u / ml (normal range 0165 u / ml). immediate cutaneous reaction to aspergillus fumigates was not available at the time of the patient s hospitalization ; nevertheless, a. fumigatus - specific serum ige levels were 1.37 u / ml. this is essential for the diagnosis of allergic bronchopulmonary aspergillosis.3 total serum igg, iga, and igm levels were quantified to rule out hypogammaglobulinemia and did not reveal any deficiencies. antinuclear and antineutrophilic cytoplasmic antibody tests were performed to exclude churg strauss syndrome, and the results were negative. there was a reduced forced vital capacity of 1200 ml (43.35% pred), reduced expiratory volume in 1 second of 720 ml (30.7% pred) with tiffeneau index, reduced expiratory volume in 1 second / forced vital capacity of 76.29, and carbon monoxide - diffusing capacity of 28%. a contrast - enhanced computed tomography (ct) scan of the chest demonstrated central cylindrical / cystic bronchiectasis involving segmental / subsegmental bronchi, bronchial wall thickening with air - fluid levels, and mucus plugging with predominant localization in the lower lobes. there was ground glass and tree - in - bud scattered in all lobes, a finding consistent with pneumonitis and acute bronchiolitis. chest ct also revealed a mosaic picture, which also emerges in all lobe findings consistent with bronchiolitis obliterans. collapse of bronchi with distal air trapping, the result of an excessively compliant bronchial wall, particularly in the left and right lower lobe, was also present (figure 2). the bronchoalveolar lavage fluid obtained from the patient for an acid - fast bacillus was negative on multiple occasions, but the same p. aeruginosa was again isolated. a diagnosis of saccular bronchiectasis due to williams campbell syndrome was made based on clinicoradiological findings, laboratory testing, and past medical history. genetic testing was planned, but the patient refused further investigations due to financial constraints. the patient was treated with intravenous antibiotics ; third - generation cephalosporins, ciprofloxacin, and acetylcysteine ; and chest physiotherapy, and was discharged after 1 month. written informed consent was obtained from the patient upon discharge for publication of this case report and all accompanying images. in 1960, williams and campbell first reported the unusual pattern of bronchiectasis in f i ve patients whose disease presented in infancy with similar clinical features. they noted markedly compliant, soft central bronchi that ballooned on inspiration and collapsed on expiration. in this syndrome, bronchiectasis due to deficiency in cartilage of the third- to sixth - order bronchi generation is observed. the syndrome is usually presented in childhood, with recurrent pneumonia and broncho - obstructive symptoms such as coughing and wheezing. nevertheless, adult cases have been reported without pathologic confirmation.2,46 the symptoms and prognosis depend on the extent of cartilage maldevelopment. ct imaging demonstrates bilateral cylindrical / cystic bronchiectasis distal to the third - generation bronchi (segmental / subsegmental) with hyperinflation of the lung.4,7 on expiration, collapse of the bronchi with distal air trapping is observed as the result of an excessively compliant bronchial wall. the trachea and central bronchi remain normal in caliber, a distinguished feature of this disease. the mechanism for the deficiency in cartilage is not well understood ; no evidence suggests that cartilage deficiency occurs outside of the bronchi.8,9 in addition, regarding diagnostic exams, bronchoscopy is often unrevealing.10 a genetic background and familial occurrence are also reported.1012 the compliant bronchi collapse during coughing, leading to poor airway drainage. subsequently, progressive obstructive disease develops, causing hyperinflation of the lung and segmental or lobar collapse. recurrent destruction of the bronchial tree and inadequate clearance of mucus result in further damage to the lung parenchyma.8,9 the long - term prognosis is variable, with rapid clinical deterioration and death in some children and prolonged survival in others.13 no specific treatment exists for williams campbell syndrome. prophylaxis from exacerbations remain the basis of treatment.14,15 prophylaxis can be achieved if an oral or intravenous antibiotic is given for 710 days or until sputum production decreases. for severe cases, several different antibiotics may be used sequentially in a continuous regimen to minimize bacterial resistance. transplantation has been reported in a patient with severe respiratory symptoms from williams campbell syndrome, but the patient died 1 year later. upon postmortem examination it was observed that the main bronchi had bronchomalacia, which was attributed to a respiratory infection during the postsurgery period.1 moreover, when necessary, based on the patient s symptoms (bronchospasm and thick, tenacious sputum), a bronchodilator, combined with postural drainage and chest percussion, can help remove secretions. respiratory exercise with free flow and bronchoscopy may be used to help mobilize secretions. hypoxia requires oxygen therapy.14 noninvasive positive airway pressure can be used when respiratory acidosis exists.16 other acquired and congenital conditions associated with bronchiectasis, including ciliary dyskinesia, cystic fibrosis, allergic bronchopulmonary aspergillosis, and immunoglobin deficiencies, must be excluded. differential diagnosis of saccular bronchiectasis includes cystic fibrosis, allergic bronchopulmonary aspergillosis, -1 antitrypsin deficiency, ig deficiency, radiation fibrosis, tuberculosis, immotile (dyskinetic) cilia syndrome, mounier - kuhn syndrome, and williams campbell syndrome. when patients signs and symptoms include recurrent respiratory infections, bronchiectasis, productive cough, and dyspnea, williams campbell syndrome should be included in the differential diagnosis.	introductionwilliams campbell syndrome, also known as bronchomalacia, is a rare disorder characterized by a deficiency of cartilage in subsegmental bronchi, leading to distal airway collapse and bronchiectasis. there have been few reports about patients affected by saccular bronchiectasis, paracicatricial emphysema, and diminished cartilage. these are all characteristic of williams campbell syndrome.case presentationthis report presents a 57-year - old woman with progressive dyspnea, cough, sputum production, and fever. the clinical and laboratory examination revealed that the patient had a respiratory infection due to bronchiectasis caused by williams campbell syndrome, which was undiagnosed in the patient until then.conclusionalthough a rare syndrome, when patients signs and symptoms include recurrent respiratory infections, bronchiectasis, productive cough, and dyspnea, williams campbell syndrome should be included in the differential diagnosis.
alzheimer 's disease (ad) is the most common form of dementia in adults. pathologically, the hallmarks of ad are amyloid plaques and neurofibrillary tangles, associated with widespread neuronal loss. its fundamental causes and the pathological cascades leading to symptoms, however, remain poorly understood. extensive evidence supports an important role of cholesterol in the development and possibly progression of ad [13 ]. the role of other lipids, such as ceramides and related - sphingolipids, (sphingomyelins, sulfatides, and glycosphingolipids) is also emerging. they can be produced by hydrolysis of sphingomyelin (sm) via sphingomyelinases (smases) or synthesized de novo from fatty acyl coa and sphingosine. conversely, in the golgi, ceramides may be transformed into sm by the addition of phosphorylcholine. additionally, glycosyltransferases can attach sugar to ceramide, turning it into glucosylceramide or galactosylceramide (galcer), a key step in the generation of complex glycosphingolipids [4, 5 ]. ceramides are important second messenger molecules that regulate diverse cellular processes including cell growth, differentiation, and apoptosis. ceramide levels also increase in response to aging and various age - related stress factors and are directly involved in apoptotic signaling in various cell types, including neurons [68 ]. there is evidence linking sphingolipid abnormalities, app processing, and neuronal death in ad. in vitro, it has been shown that -amyloid added to cultured neurons [9, 10 ] or oligodendrocytes increase smase activity, leading to an increase in ceramide levels. additional reports have found that ceramide levels increase -amyloid synthesis [12, 13 ] and favor gamma secretase processing of app [1416 ] so that inhibition of ceramide synthesis confers protection against -amyloid. thus, it has been suggested that ceramides and -amyloid may synergize to induce neuronal death. for example, the total phospholipid and sulfatide content in ad was decreased as compared to normal [1719 ], while the ceramide and galactosylceramide levels were elevated [9, 18 ]. enhanced ceramide levels have been reported in the cerebrospinal fluid (csf) of patients with ad and changes in the activity of several key enzymes of ceramide metabolism, in gene expression of pathways associated to sphingolipid metabolism have been found in brains of ad patients [21, 22 ]. during the last years, numerous mouse transgenic lines have been created and have been screened for various aspects of ad pathology [23, 24 ]. unfortunately, very little work has been done on determining if sphingolipid content is likewise perturbed in these rodent models of ad. in one study, long - chain ceramides were shown to be elevated in presenilin 1 (ps1m146v) mouse brain and to induce apoptosis in ps1 astrocytes. in a second study, sulfatides, a class of sulfated galactocerebrosides, were found to be decreased in brain tissues from two app transgenic mice (i.e., appv717f and appsw), whereas no significant changes in the content of other sphingolipid classes including sm, galcer, and ceramides were noted. by contrast, using the new mouse mutant app / ps1knock - in line, we have recently found an early and significant increase of ceramides in the cortex of these mice, without significant changes in other sphingolipid levels. however, the app / ps1ki model is unique for its drastic neuronal loss, not observed in other animal models of ad. the discrepancy in the few data available and the lack of knowledge of sphingolipid levels in the brain of other rodent models of ad prompted us to investigate whether the sphingolipid composition is altered in the brain of two other mouse models of ad : single app and double app / ps1 transgenics. concentrations of ceramides, sm, galcer, and sulfatides were determined in three brain regions : the cortex and the hippocampus, the two brain regions typically associated with the disease, and the cerebellum, a nonvulnerable region with no a plaques. for all analyses, age - matched ps1 (amyloid free) mice as well as nontransgenic wild - type mice (wt) were used. all organic solvents were of analytical grade and came from vwr international (strasbourg, france). hptlc - plates silicagel 60, 10 10- or 10 20 cm were purchased from merck (vwr international). lipid standards (nonhydroxy fatty acid (nfa) containing ceramides, ceramides containing 2-hydroxy fatty acids (2-hfa), sphingomyelins, cerebroside sulfate (sulfatides), and galactosylceramides (a mixture containing 2-hydroxy fatty acids and nonhydroxy fatty acids) were purchased from sigma - aldrich (france). aminopropyl - bonded (lc - nh2) silica gel cartridges (100 mg matrix) were from supelco (saint quentin fallavier, france). generation and detailed neuropathological analyses of the app and the app / ps1 transgenic mice have been described earlier [29, 30 ]. in brief, these mice express human app751 with swedish and london mutations (thy1 promoter) either alone or in combination with human presenilin-1 (m146l, hmg - coa promoter). in this study, 12-month - old (n = 5) app / ps1 double transgenic, 12-month - old (n = 5) ps1 single - transgenic littermates, and 12-month - old (n = 6) nontransgenic mice as well as 24 month - old (n = 5) app single transgenic and 24-months (n = 5) nontransgenic littermates were used (generous gift of sanofi - aventis, vitry sur seine, france). app mice were analyzed at 24 months of age and app / ps1 mice were assessed at 12 months of age because they revealed comparable levels of amyloid plaques in the brain at these respective ages. all the mice used in this study were female, because a gender effect with female mice displaying more severe pathology than male has been mentioned in several studies [27, 31, 32 ]. all experiments were performed in compliance and following approval of the sanofi - aventis animal care and use committee and in accordance with standards of the guide for the care and use of laboratory animals (cnrs ilar) and with respect to french and european community rules. the mice were anesthetized with pentobarbital (40 mg / kg, ip) and sacrificed. these cerebral regions were homogenized by 10 up - and - down strokes of a prechilled teflon - glass homogenizer in 20 volumes of buffer (25 mm tris - hcl, 150 mm nacl, 1 mm edta, ph 7.4) and supplemented with 50 mm sodium fluoride, 1 mm phenylmethylsulfonyl fluoride, protease, and phosphatase inhibitor cocktails (50 l / g tissue and 10 l / ml lysis buffer, resp.). the resulting pellet was resuspended in 3 volumes of ice - cold water, altogether 1.5 ml, homogenized at 4c (1015 strokes) and then sonicated for 20 s using a sonifier (branson ultrasonics, sonifier 450, danbury, ct). total lipids from brain homogenates were prepared according to previously described procedures [27, 33 ]. the mixture was stirred overnight, and then centrifuged at 1,000 g for 10 min. the pellet was re - extracted with chloroform - methanol - water (4 : 8 : 3, v / v / v) and the two supernatants combined, evaporated to dryness. partitioning was carried out using diisopropyl ether/1-butanol/50 mm aqueous nacl (6 : 4 : 5, v / v / v) according to the method of ladish and gillard. the lipids were fractionated using solid - phase extraction on 100 mg lc - nh2 cartridges (supelco, l'isle d'abeau, france) as previously described. the eluted fractions containing, respectively, free ceramides, galactosylceramides (galcer), alkali - stable phospholipids (sm), and sulfatides were applied to hptlc plates with a linomat 5 (camag, switzerland). prior to sm analysis, the phospholipid - containing fraction was subjected to mild alkaline methanolysis (treatment with chloroform : 0.6 n naoh in methanol 1 : 1 (v / v) at room temperature for 1 h) to remove glycerophospholipids. to quantify each lipid species (ceramides, sm, galcer, and sulfatides), calibration curves were obtained by running in parallel known amounts of purified lipid standards. for free ceramides, 1520 mg of brain tissue (wet weight) the plates were developed with chloroform - methanol 50 : 3 (v / v) and visualized by charring for 10 min at 180c with 3% cupric acetate in 8% phosphoric acid solution. for sm analysis, the plates were developed with chloroform - methanol - water 60 : 35 : 8 (v / v / v). sm was visualized with sulfuric acid - cuso4-ammonium molybdate spray reagent followed by heating at 110c for 15 min. galcer and sulfatides (about 0.4 mg and 2 mg of tissue per lane, resp.) were developed in chloroform - methanol - water 65 : 25 : 4 (v / v / v), sprayed with orcinol - h2so4 reagent, and then heated at 150c for 2 min. each sphingolipid species was quantified by scanning densitometry of the plates at 396 nm for ceramides, 540 nm for galcer and sulfatides, and 750 nm for sm with the camag tlc scanner 3/wincats software system. owing to the small number of animals per group, statistical analysis of the data was performed using a nonparametric kruskall - wallis test followed by a posthoc test of dunn for multiple comparisons. for the comparison of two means, all calculations were performed using graphpad prism software 3.02 (graphpad software, inc.). in single app mice (figure 1(a)), a moderate but not significant elevation of nfa - ceramides was seen in the cortex (+ 22%), with no changes of the 2-hfa - ceramide level. conversely, in the hippocampus (figure 1(b)), the nfa - ceramide level did not differ between wt and app mice, whereas a tendency towards an increase of 2-hfa - ceramides was noted (+ 19%), although it was not significant. surprisingly, as shown in figure 1(c), the level of 2-hfa - ceramides in the cerebellum of app mice was significantly increased in comparison to the counterpart of their wt littermates (+ 50%, p <.05). conversely, the nfa - ceramides showed a slight but not significant decrease compared to wt control values. however, no difference in the total ceramide content was noticed in the cerebellum of both wt (51.35 1.45 nmol / mg tissue) and app mice (50.28 3.31 nmol / mg tissue). because the double - transgenic mouse model app / ps1 develops neuropathological features of ad earlier than the single app mice, the sphingolipid analysis was performed in the brain regions of this model in younger animals (12 months of age). as shown in figures 2(a) and 2(b), concentrations of nfa - ceramides as well as those of 2-hfa - ceramides did not differ between wild - type, ps1, and app / ps1 mice in the two disease - associated brain regions (cortex and hippocampus). in contrast to the changes of ceramide content in cerebellum of app mice, the nfa - ceramide as well as the 2-hfa - ceramide levels were unchanged in this brain region in app / ps1 mice relative to their wt controls and ps1 littermates (figure 2(c)). however, nfa - ceramide content showed a tendency towards a decrease, while 2-hfa ceramides tended to slightly increase in the cerebellum of the app / ps1 mice. it should be noted that, although cortex and hippocampus of wt mice displayed almost identical ceramide content (figures 1(a), 1(b), 2(a), and 2(b)), a relatively lower nfa - ceramide content was manifest in the cerebellum (figures 1(c) and 2(c)). in normal human brain, the ceramide levels typical ceramide profiles from either cortex, hippocampus, or cerebellum of the different transgenic mice and wild - type controls were shown in figure 3. since 2-hfa - ceramides are present in extremely low levels leading to a weak staining on the hptlc plate, densitometric scanning of the plate was shown to visualize the peak corresponding to the 2-hfa - ceramide species (figure 3). to test whether other related - sphingolipids could be altered in the brain of transgenic mice, the content of sm, galcer, and sulfatides in lipid extracts of the three examined brain regions figures 1(d)1(f) show that sphingolipids (sm, galcer, and sulfatides) did not display significant changes in both cortex, hippocampus, and cerebellum of app mice compared to the wt littermates. similarly, the levels of sm, galcer, and sulfatides did not differ among nontransgenic, ps1 and app / ps1 mice in all brain region examined (figures 2(d)2(f)). it should be noted that there are differences of sm, galcer, and sulfatide concentrations between cerebral tissues (cortex and hippocampus) and cerebellar tissues. in both models, cerebellum displayed higher levels of galcer and sulfatides than cerebral tissues. this is in accordance with cheng. who also reported a ~2-fold higher level of sulfatides in the cerebellum compared to the cortex, in two other transgenic mouse models of ad. in contrast, sm levels were almost identical in hippocampus and cerebellum, but higher than those of cerebral cortex (figures 1(d)1(f) and 2(d)2(f)). examples of hptlc profiles of sulfatides, galcer, and sm and from either cortex, hippocampus, or cerebellum were represented in figures 4(a), 4(b), and 4(c), respectively. it should be mentioned that the hptlc profiles of each sphingolipid class were similar in all examined brain regions from both mouse models. using the same methodology as in our previous work, we herein analysed the sphingolipid composition of two additional models (i.e., single app and double app / ps1 mice) in which the time - course of pathology is much closer to that seen in the majority of currently available models. the main results of this study are (1) a moderate but not significant increase of nfa - ceramides and 2-hfa - ceramides in the cortex and the hippocampus respectively, of the app mice, (2) unaltered ceramide levels in the two disease - associated brain regions from app / ps1 mice, (3) unexpected alterations of the ceramide profile in the cerebellum of app mice, a region with no a pathology, and (4) no significant changes in the other related - sphingolipids in all brain structures examined of both transgenic models. based on our results and those from the literature, we will first discuss the possibility of a relationship between neurodegeneration, toxic a accumulation, and ceramide content. for the second time, why an amyloid - free brain region such as cerebellum showed ceramide alterations is discussed. ceramides were shown to accumulate in ad human brain regions and their levels vary by disease severity suggesting that they could be indicators of ad progression [9, 18, 35 ]. similarly to what happens in human ad, we previously found that ceramides increase very early in the cortex of the app / ps1ki mouse model, preceding the neuronal loss. by contrast, our present results reveal that in single app mice, ceramide levels were not significantly altered in disease - associated brain regions (e.g., hippocampus or cortex). this is consistent with the findings of cheng. who reported no changes in ceramide content in any brain region from appsw and app transgenic mice. an important difference between these single app mouse lines and the app / ps1ki model is that the latter develops an early and massive neuronal loss which correlates with strong accumulation of intracellular a42, a aggregates, and a42 oligomers [28, 36 ]. although intraneuronal a accumulation has also been documented in various app models [29, 37, 38 ], a striking difference between the models used in the present study and the app / ps1ki mice is the nature of the a peptides which accumulate. indeed, in app / ps1ki mouse brain, extremely high levels of n - truncated ax-42 variants and abundant oligomers were detected, which closely resembles that found in ad brain. by contrast, in app mouse brain, with the same total a levels as in app / ps1ki mice, only very limited levels of a42 n - terminal truncated isovariants were detected. in the app / ps1ki mice, ax-42 is the major form accumulated with a ratio ax-42/total a close to 1. in comparison, this ratio is approximately of only 0.2 - 0.3 in the app mice, and ~0.3 - 0.4 in the double transgenic app / ps1 mice, similar to the range of values reported for a large number of other app - based transgenics [28, 29 ]. n - truncated a peptides are known to aggregate more readily and are considered to be very toxic species. thus they might play a key role in the neurotoxicity observed in the app / ps1ki model. in particular, the pyroglutamate modified n - terminal truncated form of a at position 3 (a3(pe)-42), which represents a major species in the brain of ad patients, was recently shown to induce a severe neuron loss in the tba2 mouse model, a new model expressing only n - truncated a3(pe)-42 in neurons. interestingly, there is also a coincidence of considerable amounts of a3(pe)-42 and massive neuron loss in the app / ps1ki mouse model. on the basis of these findings, it is attractive to speculate that in app / ps1ki mice, the strong accumulation of intracellular toxic forms of a induces early elevation of ceramides. conversely, other app transgenic mouse models including the app mice have been reported to show no or very low a3(pe)-42 levels. this is due to the lack of posttranslational modifications such as n - terminal degradation and pyroglutamyl formation in these mice. because app mice display neither neuronal loss nor accumulation of highly toxic a 42 isoforms, this may at least in part explain why no significant accumulation of ceramides occurred in the disease - associated brain regions of these mice. thus, despite numerous neuropathological, biochemical, and even behavioral changes representative of ad developed by these app mice [23, 2830, 33, 44 ], they may not constitute a relevant model to further explore the role of ceramides in ad pathology. however, answering the question of the relationship between neurodegeneration, toxic a accumulation, and ceramide elevation could be facilitated using restricted models either lacking (i.e., single app mice) or mimicking only some specific ad - related neuropathological alterations (i.e., tba2 mice mentioned above). indeed, owing to the simultaneous occurrence of numerous pathological features of ad, the connection between them is often difficult to unravel. we next determined the ceramide content in the double transgenic mouse model app / ps1, but in younger animals because the app / ps1 mice develop neuropathological features of ad earlier than single app mice. at 12 months of age, these double transgenics revealed comparable levels of amyloid deposits than 24-month - old app mice. our results showed that at 12 months of age, ceramide levels were unaltered in both cortex and hippocampus of app / ps1 mice in comparison to age - matched ps1 mice and wild - type controls. it should be noted that there is no neuronal loss in these mice as old as 17 months, but unfortunately, older app / ps1 mice were not available for this study. however, in our previous work, we demonstrated that elevation of ceramides occurred very early (3 months of age) in the cortex of the app / ps1ki model, preceding by far the neuronal loss detectable at 6 months of age. why the app / ps1ki mice showed an increase of ceramides several months before the appearance of neuronal death while the app / ps1 mice did not is currently unknown. one possible explanation is that the neuronal loss reported in the app / ps1 mice is moderate and more restricted than in the app / ps1ki model. indeed, the loss of neurons in the former involves only the hippocampal pyramidal cell layer (loss of ~30% in 17-month - old animals). this may in some way account for the lack of ceramide accumulation in the cortex of these mice. by comparison, in the app / ps1ki model, the cell loss is greater (~50% at 10 months of age) and has been shown to extend to other brain areas such as frontal cortex and cholinergic system. moreover, as discussed above, accumulation of n - truncated a peptides should be lesser in app / ps1 mice, since the ratio ax-42/total a is of 0.3 only, versus 1 in the app / ps1ki mice. in this context, it would seem likely that the level of highly toxic a3(pe)-42 form, which is thought to be involved in neuronal death, is reduced in the app / ps1 model. it is also possible that, at 12 months of age, it is too early to visualize an elevation of ceramides, owing to the slowest progression of ad lesions in these mice than in the app / ps1ki model. since we did not have older app / ps1 mice, we should therefore be cautious to interpret the results of ceramide composition in these mice, because we can not exclude the possibility that ceramides increase at a later age. the most unexpected finding of the present study was alteration of the ceramide composition in the cerebellum of app mice, a brain region lacking a deposits and regarded as nonvulnerable to the disease. intriguingly, we noted a significant increase of 2-hfa - ceramides (+ 50%) which was concomitant with a slight decrease in nfa - ceramides. however, considering the total ceramide content, it was almost similar between wild - type and app mice. previously, we found a more dramatic 161% increase of 2-hfa - ceramides in the cortex of app / ps1ki mice but contrary to the present results, it was accompanied by an elevation of nfa - ceramides. as evident from the literature, the current knowledge about the metabolism and physiological function of 2-hfa - ceramides is very limited. in particular in ad studies, no attention was paid to 2-hfa - ceramides, rendering it very difficult to draw conclusions about the role of these ceramide species in ad physiopathology. nevertheless, a couple of interesting facts suggest that these hfa species may participate to ad pathology : (i) it was recently found that a selectively bound to sphingolipids that contained a 2-oh group on the ceramide backbone and did not effectively interact with sphingolipids that contained a nonhydroxylated fatty acid, favoring a conformational shift that disrupts membrane stability and promotes peptide - peptide interactions and oligomer formation ; (ii) the enzyme udp - galactose : ceramide galactosyltransferase (cgt), which transfers galactose to both nfa- and 2-hfa - ceramides, was found to be upregulated in human ad brain. interestingly, overexpression of cgt in transgenic mice led to a reversal nfa : hfa - galcer ratio which resulted in both decrease in hfa - galcer and increase in nfa - galcer levels. reducing the hfa - galcer level would lead to an accumulation of their immediate precursors, 2-hfa - ceramides ; (iii) there is some evidence for enhanced apoptosis - inducing activity of 2-hfa - ceramides compared to nfa - ceramides, and this effect seems to be cell type specific. in this sight, mouse mutants with defective saposin d dramatically accumulate hfa - ceramides in cerebellum, resulting in a selective loss of cerebellar purkinje cells ; (iv) we reported a gender - specific expression of hfa- versus nfa - ceramides in the app / ps1ki mouse model of ad, and this biochemical feature could be related to the increase propensity of females to develop earlier neuronal loss. although the degree of 2-hfa - ceramide accumulation in the cerebellum of app mice is much lesser than that seen in the cortex of the app / ps1ki mice, the reasons for these biochemical changes in this amyloid - free brain area are currently unknown. possibly, it might reflect alterations of ceramide metabolism, since there is evidence that other metabolic pathways are perturbed in the cerebellum of these mice. hydroxy fa sphingolipids are synthesized by the same set of enzymes as nonhydroxy sphingolipids, except for fatty acid 2-hydroxylase (fa2h). the expression of fa2h is highly variable among cell types and is inducible by certain stimuli. one may speculate that, upon unknown signal, 2-hfa - ceramides may be preferentially synthesized instead of nfa - species. it has been shown that galactosylceramidase, which forms 2-hfa - ceramides from galcer is up - regulated, whereas acid ceramidase which hydrolyzed 2-hfa - ceramides into hfa and sphingosine is down - regulated in human ad brain. thus, with combinatorial up- and down - regulation of enzymes involved in sphingolipid metabolism, the cell could modify the levels of 2-hfa - ceramides in response to the changing cellular environment. however, this is highly speculative and further investigation is warranted to determine whether these factors or other unknown factors contribute to such changes of the ceramide profile in the app cerebellum. it should be noted that also intriguing is the substantial elevation of ceramide reported by han. in the cerebellum of ad patients, we also examined the content of other ceramide - related sphingolipid classes including sm, galcer, and sulfatides. similarly to what we observed in the app / ps1ki model, we did not found any changes of sm and galactolipid levels in all brain regions from the two transgenic lines investigated. similar findings were seen in appsw and appv717f transgenic mice, respectively, except for sulfatides. indeed, by contrast to our results, a loss of sulfatide content was observed in multiple brain regions of these animals. the reasons for such discrepancies are still unclear, but it may be ascribed to the different genetic background of mouse lines and/or the genetic constructs based on different app mutation and different promoters. supporting this, it has been shown for example, that app transgenic and wildtype mice on c57bl/6 background have lower basal cholesterol levels than the ki mouse lines which are on c57bl/6 50%-cba 25%-129sv 25% background. another example is the difference of lipid composition reported by sawamura and coworkers between mouse lines with c57bl/6j and fvb / n backgrounds, respectively. moreover, these authors found that ps2 transgenic mice with c57bl/6j background displayed significant phospholipid alterations, particularly of sm, as compared to their wild - type controls, while ps2 transgenic mice with fvb / n background did not. these few examples point out the difficulties to compare the results from various mouse lines and reinforce the idea that additional studies in this field are required. in summary, this study extends previous observations on sphingolipid alterations in animal models of ad. despite several limitations, in particular the lack of old double transgenics, the present results demonstrated that, in the absence of neurodegeneration, no elevation of ceramides occurred in disease - affected brain regions from single app mice, thus corroborating recent findings in two other single app mice. moreover, since both neuronal loss and accumulation of toxic n - truncated a peptides are lacking in the app model, this might suggest that a-induced ceramide production is an important event leading to neuronal death. in the future, to support this hypothesis, it will be interesting to analyse the sphingolipid composition of the tba2 mice, the new model expressing only n - truncated a3(pe)-42 and which develops a severe neuronal loss, to evaluate whether or not ceramides, especially the 2-oh species, accumulate in the brain of these mice. accumulating and crossing the information obtained from various animal models will help to better understand the exact mechanism by which these sphingolipids contribute to ad pathogenesis.	there is evidence linking sphingolipid abnormalities, app processing, and neuronal death in alzheimer 's disease (ad). we previously reported a strong elevation of ceramide levels in the brain of the appsl / ps1ki mouse model of ad, preceding the neuronal death. to extend these findings, we analyzed ceramide and related - sphingolipid contents in brain from two other mouse models (i.e., appsl and appsl / ps1m146l) in which the time - course of pathology is closer to that seen in most currently available models. conversely to our previous work, ceramides did not accumulate in disease - associated brain regions (cortex and hippocampus) from both models. however, the appsl / ps1ki model is unique for its drastic neuronal loss coinciding with strong accumulation of neurotoxic a isoforms, not observed in other animal models of ad. since there are neither neuronal loss nor toxic a species accumulation in appsl mice, we hypothesized that it might explain the lack of ceramide accumulation, at least in this model.
cystic lesions of the lungs encompass a wide spectrum of rare lung lesions comprising of congenital cystic adenomatoid malformation (ccam), congenital lobar emphysema (cle), bronchopulmonary sequestration, and bronchogenic cyst. ccam with an incidence of 1 in 10,000 to 25,000 pregnancies is the most common lung cyst. an abnormal antenatal ultrasound scan has now become the most common mode of presentation in europe and the united states. in a small proportion of the cases fetal intervention, like thoracoamniotic shunts, laser ablation, and resectional surgery, is a possibility in some of these conditions. management of an asymptomatic lesion is controversial ; while some advocate surgical excision, others recommend resection, only when symptoms occur. this study retrospectively reviews our experience of congenital cystic lung lesions in terms of antenatal diagnosis, etiologies, presentation, management and complications. the records of all patients managed at our center with a diagnosis of cystic lung lesions from january 2000 to june 2011 were reviewed. the following data were compiled : antenatal diagnosis, symptomatology, treatment received prior to referral to our center, imaging findings, location, and type of lesions, type of surgery, postoperative ventilation, complications, and outcomes. for the 10-year study period, 40 symptomatic patients with a diagnosis of congenital cystic lung lesions were managed at our center. only three (8%) patients were diagnosed antenatally. among the remaining, 34 had no antenatal ultrasound scan performed and it was reported as normal in three patients. out of the three patients with an antenatal diagnosis, two became symptomatic at birth and one at 48 months. twenty - nine of the 40 (73%) patients were male and 11 (27%) were female and the median age of presentation was 25 months. all patients were investigated with a standard chest x - ray (cxr) and computed tomography (ct) scan. the cxr was accurate in identifying a cystic lesion in 18 patients (45%) compared with a ct scan, which picked up the lesion in all 40 patients (100%). nineteen (47.5%) children became symptomatic in the neonatal period, 13 (32.5%) between one month and one year, seven (18%) between one and five years, and one (2%) beyond five years of age. respiratory distress was the most common presenting symptom in children less than one year of age, while respiratory infections were common in children presenting after one year of age. eight (20%) children had received a full course of prior antitubercular therapy due to misdiagnosis. twelve (30%) children had an intercostal drain (icd) inserted, prior to referral to our center, due to a mistaken diagnosis of pneumothorax. all patients underwent open surgical resection by posterolateral thoracotomy, out of which eight were performed in the neonatal period. the average duration of the postoperative icd drainage was 3.5 days (1 - 11 days) and the average duration of hospital stay was five days (4 - 11 days). the final histological diagnosis was ccam in 19 (48%), cle in 11 (28%), bronchogenic cyst in five (12%), and pulmonary sequestration in five (12%). out of 40 patients, 21 (52%) had lesions on the right side and 19 (48%) on the left side, and four patients had multilobar involvement. two ccams involved all the lobes, one right ccam involved both upper and middle lobes, and one right cle involved both middle and lower lobes [figure 1 ]. thirty patients underwent lobectomies, two underwent pneumonectomies, and eight underwent excisions for a bronchogenic cyst and for extralobar pulmonary sequestrations [table 1 ]. lobar distribution of the cystic lesions (including four patients with multilobar involvement) surgical procedures performed on the various cystic lesions complications included persistent non - expansion of the residual lobe requiring bronchoscopy and suction in two patients, pneumothorax necessitating re - icd reinsertion in one patient, persistent hyperinflation necessitating a redo thoracotomy, and excision of a small foregut duplication cyst in one patient (this patient had been operated for a cle), persistent bronchopleural fistula requiring redo thoracotomy and repair in one patient. the median period of follow up was 36 months and all patients had been asymptomatic. antenatal diagnosis of cystic lung lesions is the norm in the western setup, leading to prenatal counseling and planned delivery. the antenatally diagnosed lesions need to be evaluated postnatally with a ct scan, as the cxr may appear normal in spite of a pathological lesion. the reported sensitivity of cxr as a test for the presence of ccam is about 61%, while it is 100% for a ct scan. in our series the cxr picked up the lesions in only 18 patients (45%), whereas, the ct scan picked up all the chest lesions (100%). occasionally, a ct or magnetic resonance (mr) angiography may be required for better delineation of the vascular supply in case of pulmonary sequestration. symptomatically, neonates with cystic lesions tend to manifest with respiratory distress while the older children present with recurrent infections. out of the 23 children with respiratory distress at presentation, 21 (92%) were neonates, and out of the 27 patients with recurrent respiratory infections, only 10 (37%) were neonates. twelve (30%) patients had prior icd insertion, due to mistaken diagnosis of pneumothorax. likewise, eight (20%) patients had been misdiagnosed as tuberculosis and had received complete antitubercular therapy before referral to our center. antenatal diagnosis, coupled with an awareness of congenital cystic lesions of the lung, could have prevented this wrong treatment. surgical resection is warranted for symptomatic lesions, but treatment recommendations for asymptomatic lesions are still unclear. the main arguments for surgical resection in asymptomatic lesions are the potential risk of recurrent infections, pneumothorax, and even malignant transformation. about 4% of the pulmonary tumors the tumors that develop are, sarcomas, pleuropulmonary blastoma, bronchogenic carcinoma, and mesenchymoma. in a case report and literature review reported 33 cases of primary pulmonary rhabdomyosarcomas, 15 of which arose in a pre - existing pulmonary cystic malformation. adzick. in their large series of 105 asymptomatic patients demonstrated that surgical resection is safe with no mortality and minimal morbidity. in their review and meta - analysis of the postnatal management of cystic lesions, concluded that although the risk of asymptomatic cases developing symptoms is small (3%), elective surgery in asymptomatic cases has significantly reduced postoperative complications compared to emergency surgery in these cases. also they mentioned that segmentectomy was associated with more residual disease (15%) compared to cases in which lobectomies were carried out (0%). all our patients underwent lobectomies and none have shown evidence of residual disease on follow up. minimally invasive surgery has emerged as the standard of care for many pediatric surgical conditions. the stated advantages are : better postoperative pain control, shorter hospital stay, and improved cosmesis, compared to standard thoracotomy. thoracoscopic resection of ccam has been reported to have a longer operative time, shorter hospital stay, and potentially reduced complications, with no additional costs, with the main risk factor for conversion to thoracotomy being a prior history of pneumonia. all our patients underwent posterolateral thoracotomy and resection, with an average hospital stay of five days. probably with the increasing use of video - assisted thoracoscopic surgery (vats), thoracoscopic resection of cystic lesions although the treatment of cystic lung lesions is quite straightforward, lesions that are not diagnosed antenatally are quite a diagnostic challenge when they become symptomatic, especially in our country (india), where pediatric surgical facilities are not readily available. increased awareness of this uncommon entity coupled with a wider antenatal ultrasonography (usg) cover for pregnant mothers would result in prompt referrals to a pediatric surgical center, thereby, obviating unnecessary prolonged and needless medical management like icd insertion and antitubercular therapy.	background : a majority of cystic lesions in the western world are detected antenatally, whereas, the diagnosis in our setup occurs once the child becomes symptomatic. surgical management is primarily dictated by the presence of symptoms, recurrent infection, and rarely by the potential risk of malignant transformation.materials and methods : a retrospective analysis was carried out on all consecutive patients with cystic lung lesions managed at our center from january 2000 through june 2011 for antenatal diagnosis, presentation, diagnostic modalities, treatment, and complications.results:forty cystic lung lesions were identified. only 8% were antenatally detected. out of 40, the final diagnosis was congenital cystic adenomatoid malformation in 19, congenital lobar emphysema in 11, and bronchogenic cysts and pulmonary sequestration in five each. of these, 20% had received a course of prior antitubercular therapy and 30% had an intercostal drain inserted prior to referral to our center. postoperative morbidity in the form of bronchopleural fistula, pneumothorax, and non - expansion of the residual lung was noted in 10% of the patients.conclusion:antenatal diagnosis of these lesions is still uncommon in third world countries. prior to referral to a pediatric surgical center a large number of patients received antitubercular drugs and an intercostal drain insertion, due to incorrect diagnosis.
pelvic organ prolapse (pop) is part of a range of conditions that are related to pelvic floor dysfunction such as urinary incontinence, bowel disorders and the report of vaginal bulging [1, 2 ]. women have an 1111.8% chance of undergoing at least one surgical intervention for pop or incontinence by the age of 79 years, with a re - operation rate of 29.2% [4, 5 ]. complication rates after surgery in different racial groups were reported to be 19.4% (white), 34.1% (black) and 27.4% (other). although only a relatively small number of women with pop seek treatment, it is expected that this number will increase in the future. at present, the direct cost of pop surgery already exceeds 1 $ billion per year in the united states alone. to estimate the care requirements of women with pop in the future, it is important to have reliable prevalence data from a general female population. data must be obtained using a questionnaire and vaginal examination because there is only a moderate correlation between the signs of pop (measured by vaginal examination) and the symptoms (measured by a questionnaire) [9, 10 ]. to obtain reference data on the prevalence and distribution of pop signs and the pop symptom of feeling and/or seeing a vaginal bulge, we conducted a cross - sectional study on a general population of women aged 4585 years. our first aim was to develop and validate a prediction model that will be helpful to researchers and health care policy makers. we focussed on three different cut - off points because of a discrepancy between feeling and/or seeing a vaginal bulge (the cornerstone symptom of pop) and the presence of pop signs in literature. a high correlation between signs and symptoms can be present when a different cut - off point is taken regarding the presence of pop. therefore, we looked at 1 cm above, at the hymen and 1 cm beyond the hymen. secondly, we created a pop score chart and a prognostic index to estimate the presence of pop in a general population without vaginal examination and the amount of care needed by women. a cross - sectional study was performed on a general population of women aged 45 to 85 years. 1flowchart of the study flowchart of the study the study population comprised 2,979 women, registered in the office records of eight out of nine general practitioners in brielle. brielle is a town near rotterdam (the netherlands) with 16,000 citizens. because all inhabitants have the obligation to be registered in a general practitioners clinic, the study population contained 95% of all women in brielle. names and addresses of all 2,979 eligible women aged 4585 years were obtained from the general practitioners. the women were sent information about the pelvic floor study and could be enrolled by filling out an informed consent form. they were offered three options : to sign a refusal form, or to fill out the questionnaire only or to fill out the questionnaire and undergo vaginal examination. a reminder, containing the same questionnaire, was sent 8 weeks after the first contact. non - responders were invited to complete a short questionnaire that comprised five questions about age, parity, presence of stress urinary incontinence (yes / no), faecal incontinence (yes / no) and feeling of vaginal bulging (yes / no). to encourage a high response to the questionnaire, we used envelopes with the name and logo of the erasmus university, coloured paper and stamped - addressed - return envelopes. the questionnaire used in this study combined several dutch validated pelvic floor questionnaires, such as the urogenital distress inventory and the defaecation distress inventory. in addition, subjects were asked about ethnicity, parity, vaginal bulging, incontinence, pelvic girdle pain and vaginal bulging during pregnancy, family history, menopausal status, hormone replacement therapy (hrt), previous pelvic floor surgery, educational level, smoking and heavy physical work at present or in the past. from all the participants who gave informed consent in the beginning of the study to undergo vaginal examination (n = 1,140), 800 women were randomly selected by age for popq measurement. (all response forms of the women were registered with a number that identified the age, and they were at random taken by a research assistant). a gynaecologist (mv) and a physiotherapist (ms) performed the vaginal examinations. the two examiners practiced the popq measurement protocol until they reached agreement about the test and registration scores. this process was performed at the pelvic floor centre at the erasmus medical centre in rotterdam. after each examination, all the details were entered into the three - by - three popq grid. women were assigned to one of five ordinal stages of prolapse (04) in accordance with the popq grading system. all the methods, definitions and descriptions were in line with the ics. to make a detailed analysis of stage 2, we divided it into 2a (indicating 1 cm above the hymen), 2b (0) and 2c (1 cm beyond the hymen). so, for example, the cut - off point 2a means that all subjects with pop stage 2a till stage 4 are used for analyses. within the vaginal examination group, the women were classified into the symptomatic group if they reported feeling and/or seeing vaginal bulging, and all others were included in the asymptomatic group. three different definitions of prolapse were used and compared, based on the three cut - off points on the popq scale 2a, 2b and 2c. this strategy eliminates most of the purely random variables and improves the chance that the model will perform well in future patients. the predictive performance of the three resulting models was transformed into receiver operating characteristic (roc) curves and the areas under the curves (auc) were compared. as multivariate analysis is severely hampered by missing values and more importantly, results may be biassed, we used multiple imputation with ten datasets. internal validation of the models was performed by a bootstrap re - sampling procedure : the model building process was repeated 200 times after creating 200 new datasets (bootstrap samples) by randomly drawing cases (with replacement) from the original data. the model estimates of each bootstrap sample were evaluated on the basis of the original data. on average predictions that deviate strongly from the mean usually differ greatly from the observed outcomes due to over - fitting of the model. the size of the over- fitting effect was estimated by averaging the 200 bootstrap samples. the bootstrap method was also used to estimate the amount of optimism in the auc by optimally fine - tuning a model and subsequently evaluating its predictive performance on the same data. the prediction model that showed the highest auc was translated into a pragmatic prognostic score, the slieker pop score. for each prognostic factor in the model, for ease of use, the regression coefficients were scaled and rounded to whole numbers, such that the minimum and maximum score of women in our data set were 0 and 100, respectively. from a graph the analyses were performed using the statistical package for social science (spss) 15.0. the medical ethics research committee of the erasmus medical centre in rotterdam, the netherlands, approved this study. from all the participants who gave informed consent in the beginning of the study to undergo vaginal examination (n = 1,140), 800 women were randomly selected by age for popq measurement. (all response forms of the women were registered with a number that identified the age, and they were at random taken by a research assistant). a gynaecologist (mv) and a physiotherapist (ms) performed the vaginal examinations. the two examiners practiced the popq measurement protocol until they reached agreement about the test and registration scores. this process was performed at the pelvic floor centre at the erasmus medical centre in rotterdam. after each examination, all the details were entered into the three - by - three popq grid. women were assigned to one of five ordinal stages of prolapse (04) in accordance with the popq grading system. all the methods, definitions and descriptions were in line with the ics. to make a detailed analysis of stage 2, we divided it into 2a (indicating 1 cm above the hymen), 2b (0) and 2c (1 cm beyond the hymen). so, for example, the cut - off point 2a means that all subjects with pop stage 2a till stage 4 are used for analyses. within the vaginal examination group, the women were classified into the symptomatic group if they reported feeling and/or seeing vaginal bulging, and all others were included in the asymptomatic group. three different definitions of prolapse were used and compared, based on the three cut - off points on the popq scale 2a, 2b and 2c. this strategy eliminates most of the purely random variables and improves the chance that the model will perform well in future patients. the predictive performance of the three resulting models was transformed into receiver operating characteristic (roc) curves and the areas under the curves (auc) were compared. as multivariate analysis is severely hampered by missing values and more importantly, results may be biassed, we used multiple imputation with ten datasets. internal validation of the models was performed by a bootstrap re - sampling procedure : the model building process was repeated 200 times after creating 200 new datasets (bootstrap samples) by randomly drawing cases (with replacement) from the original data. the model estimates of each bootstrap sample were evaluated on the basis of the original data. on average predictions that deviate strongly from the mean usually differ greatly from the observed outcomes due to over - fitting of the model. the size of the over- fitting effect was estimated by averaging the 200 bootstrap samples. the bootstrap method was also used to estimate the amount of optimism in the auc by optimally fine - tuning a model and subsequently evaluating its predictive performance on the same data. the prediction model that showed the highest auc was translated into a pragmatic prognostic score, the slieker pop score. for each prognostic factor in the model, for ease of use, the regression coefficients were scaled and rounded to whole numbers, such that the minimum and maximum score of women in our data set were 0 and 100, respectively. from a graph, the corresponding risk of pop can be read off. the analyses were performed using the statistical package for social science (spss) 15.0. the medical ethics research committee of the erasmus medical centre in rotterdam, the netherlands, approved this study. the response rate to the questionnaire was 62.7% (1,869 of 2,979). in the group of 1,869 responders, 472 (15.8%) women refused to participate, 1,397 (46.8% ; group 1) women agreed to fill out the large questionnaire and 1,140 (38.2% ; group 2) agreed to fill out the large questionnaire and undergo vaginal examination. in the non - responder group 3, 20.8% returned the completed short questionnaire (620 of 2,979). feeling vaginal bulging was reported by 6.7% (n = 41) of this non - responder group versus 9.8% in the responder group (135 of 1,397). from group 2, 800 out of the 1,140 women who consented to undergo vaginal examination were selected at random and sent an invitation for vaginal examination : 649 women participated (81.1%), which was 21.7% of the total study population and 46.4% of the women who filled in the questionnaire. the vaginal examination group of 649 women was stratified into an asymptomatic control group (n = 570) and a symptomatic (n = 79) group in which the women had reported seeing and/or feeling vaginal bulging. combining the data on the large and short questionnaires from the responders and the initial non - responders (1,397 + 620 = 2,017) revealed the report of a feeling of vaginal bulging prevalence rate of 8.7% (n = 176). baseline characteristics of the total study population and the different groups (group 1 the total group, vaginal examination group 2 divided into a symptomatic group and an asymptomatic group and the non - responder group 3) are shown in table 1. table 1baseline characteristics of the total study population group 1, group 2 who underwent vaginal examination divided into symptomatic and asymptomatic women expressed as percentages (%) with means and the non - responders who filled out the short - questionnaire group 3questionnaire 1, group 1 n = 1,397vaginal exam, group 2 n = 649short questionnaire non - responders, group 3 n = 620characteristics of the study populationno bulgebulgen = 570n = 79 (12.2%)mean age (range 4584) years58.0 (sd 9.2)58.0 (sd 8.9)59.3 (sd 9.1)59.2mean bmi25.6 (sd 3.9)25.6 3.725.5 3.1ethnicity white1,351 (98.4)545 (98.7)78 (100) non - white20 (1.5)7 (1.3)0educational level(n = 1,374)(n = 556)(n = 78) primary only139 (9.9)63 (11.3)7 (9) intermediate1,039 (75.6)420 (75.5)60 (76.9) higher196 (14.3)73(13.1)11 (14.1)(n = 1,340)(n = 551)(n = 78)parity, median2222 0120 (8.9)46 (8.3)3 (3.8)67 (10.6) 1215 (16)71 (12.9)13 (16.6)102 (16.1) 2675 (50.3)273 (49.5)46 (58.9)277 (43.6) 3387 (28.8)161 (29.2)16 (20.5)180 (28.3)menopausal status(n = 1,383)(n = 557)(n = 79) (pre)menopausal374 (27)151 (27.1)16 (20.2) postmenopausal1,009 (72.9)406 (72.9)63 (79.7)(pre)menopausal with hrt(n = 1,361)(n = 551)(n = 79) (pre)menopausal with hrt24 (1.7)9 (1.6)0 postmenopausal with hrt63 (4.6)23 (4.2)7 (8.9)smoking(n = 1,382)(n = 556)(n = 78) current smoker280 (20.2)117 (21)16 (20.5)(n = 228)(n = 39) ever smoker345 (46.3)158 (54.8)25 (64.1)incont in pregnancy342 (25.8)141 (30.7)23 (26)(n = 1,328)(n = 541)(n = 75)pop in pregnancy270 (20.3)113 (20.9)28 (37.3)surgical history(n = 1,384)(n = 557)(n = 79) prolapse103 (7.4)37 (6.6)16 (20.2) incontinence47 (3.4)21 (3.8)3 (3.9) hysterectomy234 (16.9)85 (15.3)20 (25.3)family history(n = 985)(n = 397)(n = 44) mother pop359 (26.4)139 (35)22 (50)(n = 870)(n = 357)(n = 41) mother ui258 (29.6)106 (29.7)16 (39)heavy physical workn = 1,381(n = 553)(n = 79) currently269 (19.3)109 (19.7)18 (22.8)n = 1,384(n = 556)(n = 79) ever619 (44.3)248 (44.6)39 (49.3) baseline characteristics of the total study population group 1, group 2 who underwent vaginal examination divided into symptomatic and asymptomatic women expressed as percentages (%) with means and the non - responders who filled out the short - questionnaire group 3 no significant differences were found between group 1 and group 3 or between the asymptomatic women and the symptomatic women in group 2. the prevalence of pop per pop stage in relation with the report of vaginal bulging in our general population is presented in table 2. the overall prevalence of stage 2b (all the women with stages 2b, 3 and 4) was 17.5% (114 of 649), of whom 30.7% (35 of 79) had symptoms of vaginal bulging (n = 35). table 2the prevalence of pop stage in relation to the report of vaginal bulging in percentage (n) ; pop data were missing in six women ; vaginal bulging question had not been answered by ten women)vaginal bulgingsymptomatic n = 79asymptomatic n = 570stage 015.6 (12)26.3 (146)stage 120.8 (16)39 (217)stage 2a18.1 (14)17.8 (99)stage 2b16.9 (13)10.1 (56)stage 2c7.8 (6)3.7 (6)stage 316.9 (13)3.1 (17)stage 43.9 (3)0 the prevalence of pop stage in relation to the report of vaginal bulging in percentage (n) ; pop data were missing in six women ; vaginal bulging question had not been answered by ten women) the results of the multivariate analyses on pop stages 2a, 2b and 2c are shown in table 3. significantly higher odds ratios were found especially in stages 2b (at the hymen) and 2c (beyond the hymen) for the report of vaginal bulging (3.80 and 5.47, resp.), for ageing (1.04 and 1.04, resp.), parity of 2 (2.84 and 3.06, resp.), parity of 3 (2.63 and 3.33, resp.), and pop in the mother (1.96 and 2.00, resp.). the roc curve in fig. 2 shows that the largest auc were 0.759 for beyond the hymen and 0.723 for at or beyond the hymen. the auc values were corrected for optimism 0.672 and 0.648, respectively. table 3results of the multivariate logistic regression analysis with test scores and area under the curve (auc) in pop substages 2a, 2b and 2c in relation to the hymen (pregn. pop = vaginal bulging symptoms during pregnancy with at least a little bother)distance 1, 2adistance 0, 2bdistance 1, 2cor95% cior95% cior95% civaginal bulging3.055.131.813.806.532.225.4710.452.97age (years)1.041.011.061.041.011.08bmi0.940.871.02nulliparous1.160.314.301 child0.440.171.1700>1,0002 children1.560.902.692.841.286.303.060.979.703 children1.540.852.782.631.136.113.331.0011.00postmenopausal status1.290.861.94smoking current0.520.330.820.620.351.090.570.251.31inc surgery2.230.925.412.110.835.37educ level intermediate0.670.391.14heavy work current1.320.852.040.560.922.661.530.713.35heavy work past1.710.883.32pelvic girdle pain0.540.191.55pregn. 2receiver operating characteristics of the multivariate analysis with the area under the curve of the stages 2a, 2b and 2c results of the multivariate logistic regression analysis with test scores and area under the curve (auc) in pop substages 2a, 2b and 2c in relation to the hymen (pregn. pop = vaginal bulging symptoms during pregnancy with at least a little bother) significance level 0.30 receiver operating characteristics of the multivariate analysis with the area under the curve of the stages 2a, 2b and 2c due to small sample sizes, no ors could be calculated in the multivariate analyses on hrt, hysterectomy, incontinence during pregnancy and incontinence in the mother. in table 4, the score chart is based on pop stage 2b (and 2c), i.e. pop at or beyond the hymen. after filling in the numbers on the score chart, the total score can be interpreted on the prognostic curve and will give the risk for the presence of pop in percentages. a shrinkage factor of 0.63, estimated from the bootstrap validation procedure, was applied to this model to enable optimal predictive performance in new subjects. table 4the slieker - pop - score chart and the prognostic index to read the sum score pop scoreseeing / feeling bulgeyesno score240age4549505455596064656970747579808485 score03691316192225children0123 score031917smokingyesno score08incontinence surgeryyesno score140current heavy workyesno score80pop symptoms gestationyesno score60mother with popyesno score120prognostic index (sum score) the slieker - pop - score chart and the prognostic index to read the sum score the response rate to the questionnaire was 62.7% (1,869 of 2,979). in the group of 1,869 responders, 472 (15.8%) women refused to participate, 1,397 (46.8% ; group 1) women agreed to fill out the large questionnaire and 1,140 (38.2% ; group 2) agreed to fill out the large questionnaire and undergo vaginal examination. in the non - responder group 3, 20.8% returned the completed short questionnaire (620 of 2,979). feeling vaginal bulging was reported by 6.7% (n = 41) of this non - responder group versus 9.8% in the responder group (135 of 1,397). from group 2, 800 out of the 1,140 women who consented to undergo vaginal examination were selected at random and sent an invitation for vaginal examination : 649 women participated (81.1%), which was 21.7% of the total study population and 46.4% of the women who filled in the questionnaire. the vaginal examination group of 649 women was stratified into an asymptomatic control group (n = 570) and a symptomatic (n = 79) group in which the women had reported seeing and/or feeling vaginal bulging. combining the data on the large and short questionnaires from the responders and the initial non - responders (1,397 + 620 = 2,017) revealed the report of a feeling of vaginal bulging prevalence rate of 8.7% (n = 176). baseline characteristics of the total study population and the different groups (group 1 the total group, vaginal examination group 2 divided into a symptomatic group and an asymptomatic group and the non - responder group 3) are shown in table 1. table 1baseline characteristics of the total study population group 1, group 2 who underwent vaginal examination divided into symptomatic and asymptomatic women expressed as percentages (%) with means and the non - responders who filled out the short - questionnaire group 3questionnaire 1, group 1 n = 1,397vaginal exam, group 2 n = 649short questionnaire non - responders, group 3 n = 620characteristics of the study populationno bulgebulgen = 570n = 79 (12.2%)mean age (range 4584) years58.0 (sd 9.2)58.0 (sd 8.9)59.3 (sd 9.1)59.2mean bmi25.6 (sd 3.9)25.6 3.725.5 3.1ethnicity white1,351 (98.4)545 (98.7)78 (100) non - white20 (1.5)7 (1.3)0educational level(n = 1,374)(n = 556)(n = 78) primary only139 (9.9)63 (11.3)7 (9) intermediate1,039 (75.6)420 (75.5)60 (76.9) higher196 (14.3)73(13.1)11 (14.1)(n = 1,340)(n = 551)(n = 78)parity, median2222 0120 (8.9)46 (8.3)3 (3.8)67 (10.6) 1215 (16)71 (12.9)13 (16.6)102 (16.1) 2675 (50.3)273 (49.5)46 (58.9)277 (43.6) 3387 (28.8)161 (29.2)16 (20.5)180 (28.3)menopausal status(n = 1,383)(n = 557)(n = 79) (pre)menopausal374 (27)151 (27.1)16 (20.2) postmenopausal1,009 (72.9)406 (72.9)63 (79.7)(pre)menopausal with hrt(n = 1,361)(n = 551)(n = 79) (pre)menopausal with hrt24 (1.7)9 (1.6)0 postmenopausal with hrt63 (4.6)23 (4.2)7 (8.9)smoking(n = 1,382)(n = 556)(n = 78) current smoker280 (20.2)117 (21)16 (20.5)(n = 228)(n = 39) ever smoker345 (46.3)158 (54.8)25 (64.1)incont in pregnancy342 (25.8)141 (30.7)23 (26)(n = 1,328)(n = 541)(n = 75)pop in pregnancy270 (20.3)113 (20.9)28 (37.3)surgical history(n = 1,384)(n = 557)(n = 79) prolapse103 (7.4)37 (6.6)16 (20.2) incontinence47 (3.4)21 (3.8)3 (3.9) hysterectomy234 (16.9)85 (15.3)20 (25.3)family history(n = 985)(n = 397)(n = 44) mother pop359 (26.4)139 (35)22 (50)(n = 870)(n = 357)(n = 41) mother ui258 (29.6)106 (29.7)16 (39)heavy physical workn = 1,381(n = 553)(n = 79) currently269 (19.3)109 (19.7)18 (22.8)n = 1,384(n = 556)(n = 79) ever619 (44.3)248 (44.6)39 (49.3) baseline characteristics of the total study population group 1, group 2 who underwent vaginal examination divided into symptomatic and asymptomatic women expressed as percentages (%) with means and the non - responders who filled out the short - questionnaire group 3 no significant differences were found between group 1 and group 3 or between the asymptomatic women and the symptomatic women in group 2. the prevalence of pop per pop stage in relation with the report of vaginal bulging in our general population is presented in table 2. the overall prevalence of stage 2b (all the women with stages 2b, 3 and 4) was 17.5% (114 of 649), of whom 30.7% (35 of 79) had symptoms of vaginal bulging (n = 35). table 2the prevalence of pop stage in relation to the report of vaginal bulging in percentage (n) ; pop data were missing in six women ; vaginal bulging question had not been answered by ten women)vaginal bulgingsymptomatic n = 79asymptomatic n = 570stage 015.6 (12)26.3 (146)stage 120.8 (16)39 (217)stage 2a18.1 (14)17.8 (99)stage 2b16.9 (13)10.1 (56)stage 2c7.8 (6)3.7 (6)stage 316.9 (13)3.1 (17)stage 43.9 (3)0 the prevalence of pop stage in relation to the report of vaginal bulging in percentage (n) ; pop data were missing in six women ; vaginal bulging question had not been answered by ten women) the results of the multivariate analyses on pop stages 2a, 2b and 2c are shown in table 3. significantly higher odds ratios were found especially in stages 2b (at the hymen) and 2c (beyond the hymen) for the report of vaginal bulging (3.80 and 5.47, resp.), for ageing (1.04 and 1.04, resp.), parity of 2 (2.84 and 3.06, resp.), parity of 3 (2.63 and 3.33, resp.), and pop in the mother (1.96 and 2.00, resp.). 2 shows that the largest auc were 0.759 for beyond the hymen and 0.723 for at or beyond the hymen. the auc values were corrected for optimism 0.672 and 0.648, respectively. table 3results of the multivariate logistic regression analysis with test scores and area under the curve (auc) in pop substages 2a, 2b and 2c in relation to the hymen (pregn. pop = vaginal bulging symptoms during pregnancy with at least a little bother)distance 1, 2adistance 0, 2bdistance 1, 2cor95% cior95% cior95% civaginal bulging3.055.131.813.806.532.225.4710.452.97age (years)1.041.011.061.041.011.08bmi0.940.871.02nulliparous1.160.314.301 child0.440.171.1700>1,0002 children1.560.902.692.841.286.303.060.979.703 children1.540.852.782.631.136.113.331.0011.00postmenopausal status1.290.861.94smoking current0.520.330.820.620.351.090.570.251.31inc surgery2.230.925.412.110.835.37educ level intermediate0.670.391.14heavy work current1.320.852.040.560.922.661.530.713.35heavy work past1.710.883.32pelvic girdle pain0.540.191.55pregn. 2receiver operating characteristics of the multivariate analysis with the area under the curve of the stages 2a, 2b and 2c results of the multivariate logistic regression analysis with test scores and area under the curve (auc) in pop substages 2a, 2b and 2c in relation to the hymen (pregn. pop = vaginal bulging symptoms during pregnancy with at least a little bother) significance level 0.30 receiver operating characteristics of the multivariate analysis with the area under the curve of the stages 2a, 2b and 2c due to small sample sizes, no ors could be calculated in the multivariate analyses on hrt, hysterectomy, incontinence during pregnancy and incontinence in the mother. in table 4, the slieker - pop - score - chart and pop prognostic index are presented. the score chart is based on pop stage 2b (and 2c), i.e. pop at or beyond the hymen. after filling in the numbers on the score chart, the total score can be interpreted on the prognostic curve and will give the risk for the presence of pop in percentages. a shrinkage factor of 0.63, estimated from the bootstrap validation procedure, table 4the slieker - pop - score chart and the prognostic index to read the sum score pop scoreseeing / feeling bulgeyesno score240age4549505455596064656970747579808485 score03691316192225children0123 score031917smokingyesno score08incontinence surgeryyesno score140current heavy workyesno score80pop symptoms gestationyesno score60mother with popyesno score120prognostic index (sum score) the slieker - pop - score chart and the prognostic index to read the sum score the present study was designed to investigate the prevalence of pop in a general female population and to develop a prediction model based on prognostic factors that could be considered into a prognostic index. the distribution of pelvic organ prolapse in this population indicated that pop was present at or beyond the hymen (stage 2b) in 21% of the women. within this 21%, 45% of the women had reported the symptom of seeing and/or feeling vaginal bulging. if stage 2a had been included, the prevalence would have increased to 36.4%, which is in line with the study by gutman.. our results are also comparable with those reported in many other studies, but as yet, no reliable explanation has been put forward for the discrepancy between pop stage and symptoms of vaginal bulging [3, 9, 1921 ]. explanations for the discrepancies between pop signs and pop symptoms might lie in the personal sphere, such as coping strategies, attitudes and beliefs, or in the social and economic circumstances, such as quality of life. we recommend that future research focuses on these personal and socio - economic factors in relation with pop. another possible underlying factor in the lack of conformity between the report of the symptom of vaginal bulging with the signs of pop stage is the reliability of the popq measurement. results can be influenced by the level of fullness of the bladder and bowel and by reluctance to make a maximum valsalva manoeuvre on command during vaginal examination. our results are in contrast with a recent study by kluivers. who were able to distinguish symptomatic women with clinically relevant pop from asymptomatic women without any clinically relevant pop. these differences in outcome can be explained on the basis of population selection in the study by kluivers. because women were included who were seeking treatment for one or more pelvic floor disorders at a pelvic floor centre. we developed a prediction model that has substantial sensitivity and specificity to help researchers estimate the prevalence of clinically relevant pop on a basis of a short questionnaire alone. to diagnose pop symptoms, we focused on the report of feeling and/or seeing vaginal bulging, as cornerstone of the symptom of pop, because in the literature, other variables such as urinary splinting, digital manipulation, defaecation disorders and pelvic heaviness show no correlations with the presence of pop [21, 23, 24 ]. in our study, the auc was analysed based on risk factors presented in an earlier study, such as age, bmi, parity, menopausal status and hrt, smoking, hysterectomy, incontinence surgery, education level, heavy physical work currently or in the past, pelvic girdle pain, incontinence and/or the report of vaginal bulging during pregnancy and incontinence or pop in the mother. highest sensitivity and specificity were reached using the 2c score beyond the hymen (auc 0.759). however, the differences between the auc of the cut - off points at and beyond the hymen are small. this indicates that no higher correlation is present between signs and symptoms using these cut - off points. especially, the report of vaginal bulging, age, parity 2 and pop in the mother contributed to the sensitivity and specificity of this prediction model. however, we recommend the use of at or beyond the hymen as the cut - off point during pop examination instead of beyond the hymen. although the auc was lower (0.723), the difference was only small, but the advantage could be the early detection of pop. this approach might also enhance preventive strategies for more advanced pop stages [2629 ]. our findings are in line with barber. who studied the prevalence of clinically relevant pop (at or beyond the hymen) in what they referred to as a low risk population, which is also applicable to a general population. higher auc scores (of 0.90) were recently demonstrated in the study by robinson. with an artificial neural network in which 20 variables made the largest contributions to the prediction model, such as age, gravidity, parity, number of vaginal deliveries, weight of the largest vaginal delivery, bmi, menstrual status, number of years postmenopausal, race, history of chronic disease, hypertension, diabetes, chronic obstructive pulmonary disease, prior hysterectomy, prior prolapse or incontinence surgery and the use of anti - hypertensive s. in contrast, our study included family history, smoking behaviour, education, heavy physical work, pelvic girdle pain and pop symptoms during pregnancy. furthermore, the definition of pop was different in the study by robinson. the location of stages 2b and 2c at or beyond the hymen was not used in their artificial neural network at all. thus, the definition of pop (2 cm beyond the hymen) used by robinson. can account for the high auc score. we developed and validated a simple, inexpensive tool, the slieker - pop - score - chart, to predict the outcome of clinically relevant stage 2b (at or beyond the hymen) and 2c (beyond the hymen) pop with auc scores of 0.640 and 0.672, respectively. this simple self - diagnostic instrument can also help women to estimate the severity of their pop. awareness of the potential presence of pop will encourage to seek advice on how to deal with their symptoms, or they can be advised to consult a gynaecologist or a physiotherapist for pelvic floor training [2629 ] before surgery because in our opinion, surgery is not the only treatment option. raising this awareness can also have an adverse effect on women being more aware of bulging feelings in the vagina, but if prevention is a goal, detection is important. therefore, the slieker - pop - score - chart can not only be used by midwifes but also on internet or other informative media and can be used for research to preventive strategies. one of the strengths of this study was the use of vaginal examination in a large cross - sectional design. this large study population was a subgroup of an even larger group of 95% of all eligible women of brielle. because there was no referral of the general practitioners and women were addressed directly by mail, there was no strong bias selection of the group. another strength was that the results led to the development of a validated pop score instrument to estimate the presence of clinically relevant pop on the basis of eight questions, without the need for vaginal examination, which could also be helpful in epidemiological studies. furthermore, this is a self - report instrument that can support a woman s decision to consult a physician. a questionnaire can elicit socially desirable answers, although this was probably minimal due to the anonymity of responses to the questionnaire. it can be difficult to identify pop, as the situation can change over the course of the day, and there is considerable dependence on performing a maximum valsalva manoeuvre. although women were not selected by their physician, still, differences in outcome might have occurred because of selection bias in the population : women who experienced some pop symptoms could be more likely to agree for vaginal examination. this could cause overestimation of the real prevalence of pop. the distribution of pelvic organ prolapse in this population indicated that pop was present at or beyond the hymen (stage 2b) in 21% of the women. within this 21%, 45% of the women had reported the symptom of seeing and/or feeling vaginal bulging. if stage 2a had been included, the prevalence would have increased to 36.4%, which is in line with the study by gutman.. our results are also comparable with those reported in many other studies, but as yet, no reliable explanation has been put forward for the discrepancy between pop stage and symptoms of vaginal bulging [3, 9, 1921 ]. explanations for the discrepancies between pop signs and pop symptoms might lie in the personal sphere, such as coping strategies, attitudes and beliefs, or in the social and economic circumstances, such as quality of life. we recommend that future research focuses on these personal and socio - economic factors in relation with pop. another possible underlying factor in the lack of conformity between the report of the symptom of vaginal bulging with the signs of pop stage results can be influenced by the level of fullness of the bladder and bowel and by reluctance to make a maximum valsalva manoeuvre on command during vaginal examination. our results are in contrast with a recent study by kluivers. who were able to distinguish symptomatic women with clinically relevant pop from asymptomatic women without any clinically relevant pop. these differences in outcome can be explained on the basis of population selection in the study by kluivers. because women were included who were seeking treatment for one or more pelvic floor disorders at a pelvic floor centre. we developed a prediction model that has substantial sensitivity and specificity to help researchers estimate the prevalence of clinically relevant pop on a basis of a short questionnaire alone. to diagnose pop symptoms, we focused on the report of feeling and/or seeing vaginal bulging, as cornerstone of the symptom of pop, because in the literature, other variables such as urinary splinting, digital manipulation, defaecation disorders and pelvic heaviness show no correlations with the presence of pop [21, 23, 24 ]. in our study, the auc was analysed based on risk factors presented in an earlier study, such as age, bmi, parity, menopausal status and hrt, smoking, hysterectomy, incontinence surgery, education level, heavy physical work currently or in the past, pelvic girdle pain, incontinence and/or the report of vaginal bulging during pregnancy and incontinence or pop in the mother. highest sensitivity and specificity were reached using the 2c score beyond the hymen (auc 0.759). however, the differences between the auc of the cut - off points at and beyond the hymen are small. this indicates that no higher correlation is present between signs and symptoms using these cut - off points. especially, the report of vaginal bulging, age, parity 2 and pop in the mother contributed to the sensitivity and specificity of this prediction model. however, we recommend the use of at or beyond the hymen as the cut - off point during pop examination instead of beyond the hymen. although the auc was lower (0.723), the difference was only small, but the advantage could be the early detection of pop. this approach might also enhance preventive strategies for more advanced pop stages [2629 ]. our findings are in line with barber. who studied the prevalence of clinically relevant pop (at or beyond the hymen) in what they referred to as a low risk population, which is also applicable to a general population. higher auc scores (of 0.90) were recently demonstrated in the study by robinson. with an artificial neural network in which 20 variables made the largest contributions to the prediction model, such as age, gravidity, parity, number of vaginal deliveries, weight of the largest vaginal delivery, bmi, menstrual status, number of years postmenopausal, race, history of chronic disease, hypertension, diabetes, chronic obstructive pulmonary disease, prior hysterectomy, prior prolapse or incontinence surgery and the use of anti - hypertensive s. in contrast, our study included family history, smoking behaviour, education, heavy physical work, pelvic girdle pain and pop symptoms during pregnancy. furthermore, the definition of pop was different in the study by robinson. the location of stages 2b and 2c at or beyond the hymen was not used in their artificial neural network at all. thus, the definition of pop (2 cm beyond the hymen) used by robinson. can account for the high auc score. we developed and validated a simple, inexpensive tool, the slieker - pop - score - chart, to predict the outcome of clinically relevant stage 2b (at or beyond the hymen) and 2c (beyond the hymen) pop with auc scores of 0.640 and 0.672, respectively. this simple self - diagnostic instrument can also help women to estimate the severity of their pop. until now, only a small number of women with pop seek treatment. awareness of the potential presence of pop will encourage to seek advice on how to deal with their symptoms, or they can be advised to consult a gynaecologist or a physiotherapist for pelvic floor training [2629 ] before surgery because in our opinion, surgery is not the only treatment option. raising this awareness can also have an adverse effect on women being more aware of bulging feelings in the vagina, but if prevention is a goal, detection is important. therefore, the slieker - pop - score - chart can not only be used by midwifes but also on internet or other informative media and can be used for research to preventive strategies. one of the strengths of this study was the use of vaginal examination in a large cross - sectional design. this large study population was a subgroup of an even larger group of 95% of all eligible women of brielle. because there was no referral of the general practitioners and women were addressed directly by mail, there was no strong bias selection of the group. another strength was that the results led to the development of a validated pop score instrument to estimate the presence of clinically relevant pop on the basis of eight questions, without the need for vaginal examination, which could also be helpful in epidemiological studies. furthermore, this is a self - report instrument that can support a woman s decision to consult a physician. a questionnaire can elicit socially desirable answers, although this was probably minimal due to the anonymity of responses to the questionnaire. it can be difficult to identify pop, as the situation can change over the course of the day, and there is considerable dependence on performing a maximum valsalva manoeuvre. although women were not selected by their physician, still, differences in outcome might have occurred because of selection bias in the population : women who experienced some pop symptoms could be more likely to agree for vaginal examination. this could cause overestimation of the real prevalence of pop. the prevalence of pop, scored by the popq, at or beyond the hymen (2b) was 17.5% in the overall group. in the symptomatic group, 45.5% had pop stage 2b. with the newly developed prediction model (based on 17 questions) and the slieker pop score chart (based on eight questions), the risk of developing pop can be estimated by healthy women themselves. according to the researchers, the slieker pop score chart can be used to estimate the prevalence of pop at or beyond the hymen in a general caucasian population.	introduction and hypothesisestimation on prevalence and distribution of pelvic organ prolapse (pop) signs in a general female population is difficult. we therefore developed and validated a prediction model and prognostic instrument.methodsquestionnaires were sent to a general female population (4585 years). a random sample underwent vaginal examination for pop (popq). a prediction model was developed using multivariate analysis and validated in a subgroup of participants.resultspositive questionnaire - response rate was 46.8% (1,397 of 2,979). from the questionnaire group, 649 women were vaginally examined (46.5%). prevalence of clinically relevant pop was 21%. multivariate analysis demonstrated significantly higher odds ratios on the report of vaginal bulging, parity 2 and a mother with pop. the receiver operating characteristic curve showed areas under the curve of 0.672 and 0.640.conclusionsthe prevalence of pop at or beyond the hymen could be estimated in a general female population using our prediction model with 17 questions and our pop score chart with eight questions.
asthenopia can manifest itself through a variety of somatic or perceptive symptoms such as headache, watery, burning or itching eyes, blurred vision, eye ache, dry eye sensation, and double vision14 and frequently appears in association with activities requiring near viewing such as reading and writing whereby eye accommodative and vergence processes are more intense.5 growing computer use (desktops, tablets, and laptops) and similar electronic equipment use (smartphones, e - book readers, video games) have increased the prevalence of asthenopia.612 the consequences of asthenopia in children and adolescents are not completely known, although there are indications that it may interfere with attention and academic performance.1315 particularly in adults, computer use - related asthenopia interferes significantly, but not permanently, with working capacity.1619 there are few studies about asthenopia prevalence in elementary school students. in australia, ip studied 1,448 6-year - old children and found 12.6% asthenopia prevalence.20 asthenopia prevalence of 23.1% and 26.4% was found in two swedish studies, respectively.5,21 with regard to associated factors, two studies detected significant association among decreased visual acuity, myopia, and accommodative dysfunctions,5,21 while another study did not find associated ophthalmological anomalies in 82% of children examined with asthenopia.20 in view of the scarcity of studies on this subject, the aim of this study is to assess asthenopia prevalence and associated factors in schoolchildren aged 616. a cross - sectional study was conducted with all children attending the first to eighth grades at two public schools in the urban region of a medium - sized town in southern brazil between april and december 2012. the study population was selected based on lists provided by the schools. as inconsistencies were found in the lists, homes were visited, and contact was made by telephone to identify children who were still on the lists but who were in fact studying at other schools. taking estimated asthenopia prevalence to be 20%, 3 percentage point accuracy, and a 95% confidence interval (ci), a study population of 970 children was sufficient to study outcome prevalence. with regard to the analysis of associated factors, the study population was also sufficient to estimate risks of around 2.0 with minimum statistical power of 80% and a 95% ci, considering an exposed / unexposed ratio ranging from 13:1 (use of glasses) to 1:2 (internet at home). asthenopia was considered to be an outcome in situations in which a child reported having had tired and/or heavy eyes during the last week. one questionnaire related to socioeconomic and cultural issues was answered by parents or legal guardians. the other questionnaire related to the presence of asthenopia was answered by the children (figure s1). the children underwent a complete examination of visual functions, including the measurement of visual acuity, refraction test, cover test, stereopsis, heterophoria assessment, near point of convergence (npc), and accommodative convergence / accommodation (ac / a) ratio. visual acuity and refractive status were assessed using the logmar chart at a distance of 4 m for each eye separately. correction was maintained in the case of children having hyperopia equal to or greater than 1.25d, myopia equal to or greater than 0.50d, and astigmatism equal to or greater than 0.75d. the cover test was performed on all children who achieved 20/25 visual acuity in both eyes after the refraction examination. for this study, children not achieving visual acuity of 20/25 in both eyes with the best possible correction did not undergo accommodative and binocular exams and were excluded. children with anisometropia, but with visual acuity of 20/25 or better in both eyes with or without correction, remained in the study. stereopsis was assessed using the titmus test in all children who did not have tropias. howell s near and far test with a 6d prism (lower base) was used in the right eye to measure horizontal heterophorias, and a + 1.00d flipper lens was used to measure the ac / a ratio (cutoff : 4:1).22 thorington s test was used to measure vertical heterophorias.23 for the heterophorias, the cutoff point chosen were esophoria (far and close) : 0 prismatic diopters (pd), exophoria (far) 2 pd, exophoria (close) 3 pd, and vertical heterophoria 0 pd. npc was assessed using the krimsky test and luminous focus with a transilluminator, where 6 cm was the cutoff (break) point chosen.24 donders push - up test was also performed during the study period. a third orthoptic technician thus measured accommodative amplitude using the same technique in an open space in 10% of the sample as a quality control measure. cycloplegia was performed using cyclopentolate 1% eye drops for a more accurate measurement of ametropias. direct photomotor reflex and pupil size were observed after 20 minutes. in cases of photoreactive pupils or those with a diameter 6 mm, a third drop was instilled in both eyes. after a further 15 minutes, signs of pupil dilation cycloplegic autorefraction was performed using a potec auto refractor (model prk-5000, potec co., ltd. auto refractor calibration was checked at the beginning of each working day using a 5.25d eye model. once children had been aligned with the device, eight measurements were taken of each eye. the eight measurements for each eye and their mean values were obtained through thermal printing. the refractive examination and cycloplegic eye drop installation were performed by two trained orthoptic technicians supervised by an ophthalmologist. the demographic variables studied were sex (male or female), age (in completed years), and skin color (white and non - white). economic status was classified according to the criteria of the brazilian association of survey companies (associao brasileira de empresas de pesquisa).25 as this association s criterion for data on the level of schooling of the head of the family was not available in this study, it was replaced by the level of maternal schooling. with regard to environmental / behavioral variables, the time at which the child went to bed was dichotomized as regular (always at the same time, with 8 hours of sleep) and irregular, and the children were asked whether or not their families had a computer / internet and video games at home. parents were also asked whether or not their children had video games or internet at home, but we did not estimate the time of use and whether or not they wore glasses. refractive status was categorized into (a) uncorrected emmetropic, hyperopic, or astigmatic at 6 cm in 14.0% of the children, and the ac / a ratio was found to be altered in 17.1% of them (table 1). at the univariate analysis stage, age was directly associated with asthenopia and accounted for risks in 51% of those aged 1014 and in 69% of those aged 1516 compared with children in the age range of 69 years. video games, computers, not going to bed on time, acuity below 20/25, the presence of ametropias, near vertical heterophoria, near horizontal heterophoria, far horizontal heterophoria, ac / a ratio, normal npc, and stereopsis were not associated with asthenopia (table 1). with regard to the adjusted analysis, age was directly associated with asthenopia, and subjects aged 1516 were 1.69 times more likely to have asthenopia than those aged 69 (p=0.001). after adjusted analysis wearing glasses represented a 48% risk of asthenopia (table 2). asthenopia was associated with other eye symptoms such as burning, aching, and itching, emphasizing wide - ranging variability and the possibility of overlapping asthenopia symptoms (14) (table 1). asthenopia prevalence found in this study was similar to that found in a study conducted with 6- to 16-year - old schoolchildren in sweden (23.1%),5 but the questionnaires were not the same. in the swedish study, the questions used to define asthenopia were directed toward the group of symptoms relating to near visual effort. in australia, 12.6% of the 6-year - old children had asthenopia - related complaints, but the difference in prevalence may be due to the difference in the age group assessed (not primed to interpret the symptoms) and to the fact that the questionnaire was answered by those legally responsible for the children.20 another study performed in sweden with children aged 610 found 34.7% asthenopia prevalence, although as a convenience sample was used prevalence may have been overestimated. ocular complaints even tired and heavy eyes in very young children are very inaccurate, but when present, or if associated with learning disabilities perceived by parents and teachers, eye examinations are very important.21 since most studies showed no important relationship between asthenopia and visual acuity, screening only children with visual impairment would not detect a significant proportion of children with asthenopia. the combined frequency of asthenopia was 19.7% in a recent systematic review and meta - analysis of population - based prevalence studies, and the relation between asthenopia and visual acuity, binocular dysfunctions, or refraction abnormalities was controversial.28 increased asthenopia as age increases is in keeping with the literature.21 according to scheiman and wick, with effect from the fourth year of elementary education (at approximately 10 years old), children increase significantly the quantity and the difficulty of cognitive tasks and spend more time concentrating to learn.29 this increases the vergence and accommodative requirements directly related to asthenopia symptoms when reading and writing. symptoms can therefore increase if children have an accommodative and/or binocular dysfunction, such as accommodative insufficiency.5 however, the lower prevalence found in younger children may be underestimated, in part, owing to the difficulty in their understanding the questions about asthenopia, as well as a possible bias created by those wishing to give the right answer to please the interviewer.1315,3032 the association between wearing glasses and the presence of asthenopia is in accordance with an australian population - based study with 6-year - old children, in which those with asthenopia symptoms were seven times more likely to be using glasses compared with those who did not complain of having asthenopia - related symptoms, odds ratio = 7.1 95% ci (4.610.9).20 this association may be due to reverse causality, given that asthenopia symptoms are frequently one of the criteria used by eye care professionals to prescribe glasses. no association was found between asthenopia and sex, skin color, or economic status. lack of association with sex was also found in the australian population - based study with 6-year - old children (p=0.39).20 the lack of association between an altered visual function examination and asthenopia reinforces the findings of the majority of studies.1315,33,34 this aspect may be related in part to children not finishing activities that induce eye discomfort symptoms, that is, children who due to an undiagnosed visual function alteration feel discomfort when doing near activities requiring binocular, stereoscopic, and clear focus vision, naturally avoid reading, and, as a consequence, complain less about asthenopia. moreover, many children do not report having asthenopia symptoms to their parents and teachers, principally because they are not aware of what it feels like to read comfortably. this can, in part, explain the lack of association related to npc and ac / c ratio findings. two studies found association between accommodative inflexibility and accommodative insufficiency and asthenopia.5,21 integrative analysis of these findings was not remembered in our study, it not being possible to classify the children in relation to specific accommodative and binocular dysfunctions. two other studies indicated a direct relationship between asthenopia and myopia, astigmatism,5 and hyperopia greater than 3d.20 strabismus was also not associated with asthenopia, although in studies in which this association was significant, the possibility of part of the effect being due to high hyperopia (accommodative strabismus) must be remembered. the association between asthenopia and the use of computers and other electronic devices has not been established among children. the use of video games or computers at home showed no association with asthenopia, although these activities were not quantified as to the amount of time spent doing them. this can underestimate the lack of association between eletronic devices and eyestrain. in both schools, children did not use computers in the classroom.1,3,6,18,3539 analysis of the consistency of the data is hampered by the variability in the methods used by the different studies. some studies use a small sample size.21 in the literature, the definition of a child with asthenopia varied a great deal between the studies. a further difficulty with regard to comparing studies is the lack of a standardized questionnaire or an objective gold standard instrument for assessing asthenopia. in all population - based studies, asthenopia is self - reported by questionnaire and is subject to self - reporting bias having limited value due to the subjective nature of the outcome measure.28 ip used the sydney myopia study questionnaire with more than 175 general questions and just 2 relating to complaints of eye ache or tiredness.40 the visual analogue scale was used by abdi on which students locate the intensity of their visual fatigue.5 other questionnaires take into consideration characteristics associated with asthenopia but are used as an instrument for measuring symptom frequency before and after treatment for specific dysfunctions, such as the convergence insufficiency symptom survey41 and the college of optometrists in vision development quality of life outcomes assessment.42,43 none of these instruments has been validated in brazil. because of this, we used similar questions to those adopted in the literature for parents and children with good correlation. this study shows that schoolchildren have expressive visual fatigue prevalence, that the lack of association with refractive, binocular, or accommodative problems, and that the complaint increases with age. the limitations were related to the method of assessment of asthenopia (questionnaire) and the adopted cutoff break points that were derived from adult studies. complaints increase in older children as the amount of learning activities, time spent concentrating, and cognitive maturity also increase. using computers or video games as a leisure activity was not found to be associated, but the time spent on these activities was not quantified. the prevalence of visual function alterations does not differ from the general population, and, therefore, they are not prerequisites for the onset of asthenopia. considering that asthenopia is prevalent but little studied among children, it is important that its mechanisms and risk factors be better defined. in addition, health professionals need to be on the lookout for complaints of visual fatigue because of its potential to influence learning and school performance.	objectiveto assess asthenopia prevalence and associated factors in schoolchildren aged 616.methodsthis was a cross - sectional study of all children attending the first to eighth grades at two public schools in the urban region of a medium - sized town in southern brazil between april and december 2012. a questionnaire on socioeconomic and cultural matters was answered by parents, while the children answered a questionnaire on asthenopia - related symptoms. the children underwent a complete visual function examination, including measurement of visual acuity, refraction test, cover test, stereopsis, heterophoria assessment, near point of convergence, and accommodative convergence / accommodation ratio.resultsasthenopia prevalence was 24.7% in a total sample of 964 children. visual acuity of 20/25 or better in both eyes was found in 92.8% of the children. the stereopsis test was normal in 99.4% of them, and some kind of strabismus was found in 3.5%. about 37.8% had astigmatism, 71.6% had mild hyperopia, 13.6% had moderate hyperopia, and 6.1% were myopic. near point of convergence was abnormal in 14.0% of the children, and the accommodative convergence / accommodation ratio was found to be altered in 17.1% of them.conclusionchildren and adolescents have expressive prevalence of asthenopia. the prevalence of visual function alterations does not differ from the general population, and, therefore, they are not prerequisites. it is very important that its mechanisms and risk factors be better defined. health professionals need to be on the lookout for complaints of visual fatigue because of its potential to influence learning and school performance.
the kidney is thought to play an important role in the inactivation of endogenous gastrin. this is based on the tacts that hypergastrinemia is frequently observed in patients with acute and chronic renal failure and in experimental animals with bilateral nephrectomy, and the fact that the enzyme which deamide the c - terminal tetrapeptide of gastrin can be extracted from mouse kidney homogenates. we conducted this study to investigate the changes of serum gastrin concentrations in patients with renal failure and to delineate the role of gastrin on the pathogenesis of peptic ulcer that frequently combines in patients with renal failure. we also investigated not only the changes of fasting serum gastrin concentrations, but also postprandial gastrin concentations in pre- and post dialysis state. fifteen patients with renal failure who had been admitted to the department of internal medicine of kyung hee university hospital from june, 1983 to october, 1983, were studied. the control was 15 normal persons without renal disease, peptic ulcer or atrophic gastritis. after a 10-hr fasting, venous blood samples were taken and then each subject ate a standard meal. the meal consisted of two hard boiled eggs, one piece of bread and a cup of milk. postprandial blood samples were taken at 30, 60 and 120 minutes after eating the standard meal. there was no difference in age and sex between controls and the patients with renal failure. mean concentrations of serum creatinine and bun were 10.6mg% and 70.8mg% respectively in patients with renal failure in contrast to normal values in controls. of the patients with renal failure, 8 of 15 patients complained of dyspepsia and 4 of 15 had peptic ulcers (2 with gastric ulcer and 2 with duodenal ulcer). eight patients had been treated with hemodialysis (table 2). in patients with renal failure, the mean fasting serum gastrin concentration was 258.2pg / ml, a significantly higher value than that in the control (85pg / ml) (p<0.001) (table 3). the correlation coefficient between fasting serum gastrin concentrations and creatinine was 0.37, not significant statistically. meal stimulated serum gastrin concentrations were significantly higher in the renal failure group than those of control at 30, 60 and 120 minutes of postprandial samples (table 3) (p<0.001). the peak increment in gastrin concentrations was also significantly higher in the renal failure group than controls at postprandial 60 and 120 minutes samples (fig. 3) (p<0.001). there was no statistically significant difference in fasting and postprandial serum gastrin concentrations between the group of the patients with peptic ulcer and those without ulcer. and there was also no statistically significant difference in the peak increment (peak concentration- basal concentration) of gastrin between the ulcer and nonulcer group in patients with renal failure (table 4, fig. 4). in 8 patients treated by hemodialysis, when serum gastrin concentrations were measured before and after dialysis, there were no statistically significant changes due to hemodialysis with a mean predialysis value of 365.9pg / ml and a post - dialysis value of 369.8pg / ml (table 5, fig. 5). in the previous studies, it was accepted that pentagastrin was inactivated in the liver and gastrins such as g17 and g34 were inactivated in the organs other than liver. hypergastrinemia observed both in acute and chronic renal failure suggests a certain role of the kidney in metabolism of endogensous gastrin. and previous studies have shown that serum gastrin concentration parallels with glomerular function and is reduced to a normal level after kidney transplantation. but there was no change in serum gastrin concentration after hemodialysis in spite of a reduction of serum bun and creatinine. reported that a certain amount of gastrin was degraded in the renal cortex after administration of radioactive labelled gastrin. davidson. reported that about 30% of endogenous gastrin was inactivated by the kidney in one blood perfusion by measuring the difference of serum gastrin concentrations separately from the renal artery and vein. but they could nt detect any significant amount of gastrin excreted into urine, suggesting the possibility of a high rate of catabolism of gastrin in the kidney. there are two possibilities in the mechanism of hypergastrinemia in patients with renal failure ; excessive production and disturbance in catabolism of gastrin. it is accepted that renal failure per se does not induce hypergastrinemia and that accompanying atrophic gastritis or hypochlorohydria can not promote over - production of gastrin. as to the catabolism of gastrin davidson. tried to compare the changes of serum gastrin in bilateral nephrectomy and bilateral ureter ligation groups before and after development of uremia using rats as experimental animals. serum gastrin was increased only in the bilateral nephrectomy group showing that loss of normal functioning renal mass was associated with hypergastrinemia regardless of development of uremia. the present study has shown that fasting serum gastrin concentration (258.2pg / ml) was significantly higher than those of normal controls (86pg / ml) in the patients with renal failure but there was no correlation between serum creatinine and gastrin concentrations. the absence of correlation may be due to the fact that as about half of the patients had received regular hemodialysis, the serum creatinine could nt accurately reflect the degree of renal failure. but the increment of acidity in gastric juice, excessive secretion of gastrin or secondary hyperparathyroidism has been proposed. in the present study, the serum ca concentrations in the patients with renal failure were all within normal limits. however, most patients had taken phosphate binding gel for a long time and this probably made the serum ca concentrations normal. investigated the gastrin response to meal stimulation and found that this was increased and prolonged in chronic renal failure especially in the subgroup of big gastrin but not in little gastrin. they measured the clearance rate of little gastrin in 4 patients with chronic renal failure in pre- and postprandial period and reported that there were no significant changes between the two periods, suggesting that the kidney played no role in the metabolism of little gastrin. although little gastrin is 6 times more potent than big gastrin in stimulation of gastric acid secretion, as little gastrin showed no response postprandially, they suggested that hypergastrinemia did nt relate directly with the increased frequency of peptic ulceration in renal failure. in the present study, serum gastrin concentrations in patients with renal failure were significantly increased not only in fasting but also in postprandial period, more marked and prolonged in postprandial period (table 3, fig. 3). these findings are in accordance with the report of taylor. but there was no significant difference in fasting and postprandial gastrin response between the patients with renal failure with peptic ulcer and those without peptic ulcer and it is implicated that the increased incidence of peptic ulcer in renal failure could nt be related to hypergastrinemia (fig. korman. reported that there was a tendeny of reduction of serum gastrin concentrations in 8 patients with renal failure after treatment by hemodialysis, but not significant statistically. also reported that there was no significant change in serum gastrin concentrations in the patients with chronic renal failure after hemodialysis. in the present study gastrins are middle molecular weight substances and they are thought to be unable to pass through the dialysis membrane. investigated changes in serum gastrin concentrations separately in the carotid artery, jugular vein, femoral vein, renal vein and mesenteric vein of dogs during continuous intravenous infusion of little gastrin and observed that there was no significant difference in the level of gastrin concentrations among them and suggested that degradation of little gastrin could occur in all capillary endothelium. conclusively, there still remains a lot of unsolved problem in the field of study of the relationship between gastrin and renal function.	fasting and postprandial gastrin levels were measured by radioimmunoassay in serum from 15 patients with renal failure and compared with those in 15 healthy controls. pre- and posthemodialysis gastrin levels were also measured.the fasting serum gastrin levels and serum gastrin response to a standard meal in the patients with renal failure were significantly higher than those in normal controls.fasting and meal stimulated gastrin levels were not significantly different in renal failure patients with peptic ulcer when compared with those in renal failure patients without peptic ulcer.there were no statistically significant differences in the serum gastrin levels before and after hemodialysis in patients with renal failure.
teeth are exposed to a great variety of damages, which may weaken their structure. caries, fractures, attrition, and endodontic access cavities are the most common reasons of tooth structure loss rendering them susceptible to fracture. according to cavity preparation principles, it is recommended to remove all the undermined enamel to prevent functional fracture. when restoring teeth with large cavities, choosing the most ideal method and material to restore strength and esthetics is a real challenge. often it is necessary to use full or partial coverage instead of a simple intracoronal restoration. this fact has been based on clinical studies and indicates the need for special restorative considerations for teeth susceptible to fracture. effects of amalgam, gold and a variety of composite systems have been evaluated on the resistance of teeth to fracture [46 ]. wendt have shown that cast gold restorations providing cuspal protection could result in a significant increase in the resistance of teeth to fracture, compared to unaltered teeth. salis showed that gold on - lays provided good support for the remaining tooth structure. in addition, it has been revealed that in case of high stress, such teeth suffered only superficial cuspal fractures. some researchers have claimed that if the teeth are over loaded, composite restorations make the tooth fracture more promising at the tooth - resin interface [57 ]. in recent years, there has been an increased acceptance and use of bonding techniques in strengthening tooth structure [810 ]. due to the success of bonding techniques, there is an increasing interest in determining how a restoration bonded to the tooth by different methods can affect its conservation and longevity. it has been shown that the acid - etch technique might influence the deformation of a cusp under occlusal forces [1113 ]. latino found no difference between restorative materials (amalgam, bonded amalgam, and composite) regarding the ability to reinforce unsupported enamel ; however, sound dentin was the most reinforcing factor. enamel has been proved to be the most appropriate tissue against the opposing tooth enamel, both esthetically and mechanically. as the major role of the enamel is to preserve function and the natural contact points of the teeth, reinforcement of the unsupported enamel apparently, two major factors may provide the dentinal support of the enamel ; namely, strong connection at the dentino - enamel junction (dej) and appropriate dentin elasticity. inherent dentin elasticity enables it to absorb the mechanical energy applied to the tooth ; consequently, leading to resistance against masticatory forces, whereas the enamel does not possess such characteristics. needless to say that the dentino - enamel junction may serve the tooth strength by its unique characterizations. the purpose of the present study was to investigate the effect of various restorative materials and bonding agents on the unsupported enamel reinforcement and to compare it with sound dentin. in this in vitro study, forty - five extracted human molar teeth were selected, washed thoroughly, and stored in chloramine t for one week. after cleansing with rubber cup and pumice, the teeth were randomly divided into five groups of nine. high - speed hand piece and a diamond fissure bur (no=837 l 0.012, teeskavan) was used to remove the lingual cusps of each tooth. as proposed by probhakar, two cuts were used in each tooth ; the first cut was vertical, parallel to the long axis and 1 mm facial to the central groove extending mesially and distally through the marginal ridges. the second cut was horizontal, perpendicular to the long axis of the tooth on the lingual surface and approximately 2 mm coronal to the cemento - enamel junction (cej), extending to join the first cut. the group with sound dentin was considered as the positive control. in groups 2 to 5, the dentin supporting the buccal cusps was removed at approximately 3 mm gingival to the occlusal enamel of the buccal cusps using a no. all steps were carried out under sufficient light, taking care to reduce the risk of enamel crack or wall crazing, while undermining the enamel. after this step, these groups received different treatments as mentioned below : -pc, the dentin of facial cusps was left intact (positive control group).-nc, the prepared cavities were not restored (negative control group).-a, the prepared cavities were filled using two layers of varnish (kimia) and spherical amalgam (sina) inserted in 0.51 mm thick increments and thoroughly condensed. preparation and manipulation of the amalgam was performed according to manufacturer s instructions.- ba, the teeth were restored with an amalgam bonded system, (panavia, kuraray company) and spherical amalgam (sina). c, the cavities were filled using resin composite and a fifth generation dentin - bonding agent, single bond (3 m). the composite resin (tetric ceram viva dent) was inserted incrementally, each layer light cured for 10 seconds using colten light cure unit (coltolux 2.5) with light intensity of 300 mw / cm. -pc, the dentin of facial cusps was left intact (positive control group). -a, the prepared cavities were filled using two layers of varnish (kimia) and spherical amalgam (sina) inserted in 0.51 mm thick increments and thoroughly condensed. preparation and manipulation of the amalgam -ba, the teeth were restored with an amalgam bonded system, (panavia, kuraray company) and spherical amalgam (sina). -c, the cavities were filled using resin composite and a fifth generation dentin - bonding agent, single bond (3 m). the composite resin (tetric ceram viva dent) was inserted incrementally, each layer light cured for 10 seconds using colten light cure unit (coltolux 2.5) with light intensity of 300 mw / cm. all specimens were thermocycled for 1500 cycles at 3c and 45c with a dwell time of 20 seconds, and then kept at room temperature in distilled water before mounting them in self - cure acrylic resin. then a flat horizontal surface of approximately 1.52.5 mm was cut at the tips of the remaining enamel cusps using a diamond disk. all the specimens were then loaded compressively to fracture using an instron testing machine (model, 1195). the end of the rod was placed against the flattened area of the enamel (fig 1). the data were subjected to one way analysis of variance (anova) and duncan post hoc tests. the results showed that the negative control group with not restored undermined enamel was significantly weaker than all the other groups, except the composite group. the resin composite group was not significantly different from either the negative or the amalgam restored groups (p=0.056, p=0.642, respectively). bonded amalgam restorations were not significantly different from the positive group (p=0.762). both amalgam and in addition, the amalgam group revealed a significantly less reinforcing effect than the amalgam bonded restorations and the positive group (p=0.00). nowadays, bonding technology is believed to possess the capability to strengthen tooth structure [810 ]. in the present study, there was no significant difference in the amount of support provided by bonded amalgam (panavia f) and sound dentin (p=0.762), showing the ability of this method of restoration in maintenance and reinforcement of the undermined enamel. latino showed no significant difference among different restorative materials in their ability to support undermined enamel. grisanti showed no significant difference in enamel support between tooth colored restorative materials (composite resin, resin - modified glass ionomer and conventional glass - ionomer). in both the above - mentioned studies mc cullock revealed that adhesive materials increased tooth resistance to fracture as much as two to six times, depending on the applied technique. pilo proved that adhesives enhanced the reinforcement effects of bonded amalgam up to 39%61%, resulting in a recovery in amalgam stiffness. eakle also attributed fruitful results to the application of dentinal adhesive in the promotion of tooth resistance to fracture. these results support our study, which shows that amalgam bond restoration causes the same strength as sound dentin and significantly more than unrestored cusp. it should be mentioned that dias de souza reported no significant differences in fracture resistance of non - bonded amalgam combined with varnish restorations as compared with those restored with bonded amalgam plus panavia f, which is not supported by the results of the present study. molinaro showed that both packable and conventional composite resins could not provide much cuspal stiffness compared to bonded amalgam. this confirms the results of the present study, which surprisingly showed no statistically significant difference between resin composite and undermined enamel groups (p=0.056), meaning that resin composite is not able to provide satisfactory support for the undermined enamel. this does not agree with mckenzie who showed that composite restored teeth were found to be significantly stronger than those unrestored ; however, in their study, resin composite was not compared with bonded and non - bonded amalgam. other than the type of material, the method of insertion with care to prevent voids is important. bonded amalgam (panavia f) could support undermined enamel as well as sound dentin.	objective : preservation of unsupported occlusal enamel after removal of underlying carious dentin may result in maintenance of aesthetics as well as wear resistance against the opposing enamel. this study investigates the influence of different restorative materials and bonding agents on reinforcement of unsupported enamel in molars and compares it with sound dentin.materials and methods : in this in vitro study, forty- five extracted human molars were selected and randomly divided into five groups of nine. all lingual cusps were cut off. the dentin underlying the buccal cusps was removed in all groups except the positive control. the negative control group received no restorations. after application of varnish and panavia f, spherical amalgam (sina) and after application of single - bond (3 m), composite resin (tetric ceram) was used to replace missing dentin.all specimens were thermocycled, then mounted in acrylic resin using a surveyor. lingual inclination of facial cusps was positioned horizontally. load was applied by an instron machine at a crosshead speed of 10 mm / min until fracture.data were subjected to anova (one way) and post hoc test (duncan).results : statistically significant differences were found between the five groups (p<0.001) ; however, no significant difference was revealed between bonded amalgam and the positive control groups (p=0.762). composite and amalgam had the same effect (p=0.642), while the composite and negative group had no significant difference (p=0.056).conclusion : bonded amalgam systems (panavia f) could reinforce the undermined occlusal enamel effectively.
a patent foramen ovale (pfo) can act as a pathway for a thrombus from the peripheral veins, bypassing the lungs and entering the systemic circulation. systemic, noncerebral, paradoxical embolisms occur less frequently, accounting for 5%10% of all paradoxical embolisms. renal infarction secondary to paradoxical embolism has rarely been described, and paradoxical embolism involving kidneys has been reported to commonly involve multiple organs, such as the lung, kidneys, and brain. here, we report a case of a paradoxical embolism caused by pfo involving only kidneys. a 48-year - old korean man was referred to our hospital with sudden right flank pain that had begun 10 days earlier. he had been diagnosed with idiopathic thrombocytopenic purpura (itp) 10 years earlier, but his platelet counts had been stable (> 50,000/l) with no significant bleeding, over the past 10 years. he had undergone a hemorrhoidectomy two months earlier. he was a 30-pack - year smoker and an occasional drinker, with no noteworthy family medical history. on admission, the patient 's blood pressure was 107/68 mm hg and his pulse rate was 78 beats / minute. his respiratory rate and body temperature were 18 breaths / minute and 36.3 c, respectively. his neurologic findings were normal, and neither edema nor bruising was found on the lower extremities. blood test results showed a low platelet count (white blood count 6,800/l, hemoglobin 12.4 g / dl, platelet 63,000/l), and his serum creatinine was 0.83 mg / dl and blood urea nitrogen 13.0 mg / dl. urine microscopy revealed mild hematuria (red blood cell count 35/hpf) and pyuria (white blood cell count 1120/hpf). his levels of antithrombin iii activity, plasminogen activity, protein c activity, coagulation factor viii activity, homocysteine, lupus anticoagulant, and anticardiolipin antibody were within the normal ranges, suggesting no abnormality of the coagulation system. a contrast - enhanced abdominal computed tomography (ct) scan was performed in the previous hospital, showing no stones, but a wedge - shaped perfusion defect was detected in the right kidney, suggestive of renal infarction (fig. 1). contrast - enhanced abdominal magnetic resonance (mr) angiography was performed to exclude the possibility of a renal artery thrombus or dissection. because the patient 's renal arteries were intact despite acute renal infarction, the filling defect was suspected to be caused by embolization, and the cortical scars on the left kidney suggested the possibility of previous recurrent renal infraction. a duplex lower extremity vein scan showed no obvious thrombosis in the deep venous system, which is a frequent potential source of an embolism. electrocardiography showed a normal sinus rhythm, and transthoracic echocardiography revealed no thrombus, but normal left ventricular function. however, a contrast study and bubble test during transesophageal echocardiography revealed a small pfo (fig. 3a). from these findings, a paradoxical embolism through a small pfo was confirmed as the cause of the patients multifocal renal infarction. the patient had been given intravenous heparin for his renal infarction in the previous hospital. during the heparin treatment, he developed hemorrhoidal bleeding, and underwent hemorrhoid ligation and discontinued anticoagulation. therefore, pfo closure was planned to prevent further embolic events and bleeding complications, which can recur under anticoagulation therapy. the diagnosis of acute renal infarction is often missed or delayed because the disease is both rare and has a nonspecific clinical presentation. in a patient with an increased risk of thromboembolism diagnostic tests should be considered for patients who are at risk of systemic embolization and present with symptoms suggestive of renal infarct. a contrast - enhanced ct scan should be performed to look for renal infarction, for which the classic finding is a wedge - shaped perfusion defect. two major causes of renal infarction are thromboemboli, which usually originate from a thrombus in the heart or aorta and in situ thrombosis of a renal artery or renal artery dissection, which is less common. the major sources of a clot embolism include the left atrium during atrial fibrillation, a left ventricular thrombus in patients with myocardial infarction, and thromboemboli originating from complex plaques in the aorta. other potential embolic sources include valvular vegetations in patients with infective endocarditis and, rarely, paradoxical embolism through a pfo. the foramen ovale, which allows blood to flow across the atrial septum in the fetal stage, normally closes shortly after birth. however, in approximately 25% of adults, closure of the foramen ovale is incomplete. when a right - to - left shunt exists across the pfo, thrombi, and vasoactive substances formed in the right - side venous circulation can bypass the lung filter and enter the left arterial system, causing a paradoxical embolism. although the vast majority of people with pfo are asymptomatic, the presence of pfo has been implicated in stroke, migraine headache, decompression sickness, high - altitude pulmonary edema, and platypnea - orthodeoxia syndrome. although ischemic events can occur in most organs, including the brain, eyes, kidneys, spleen, and intestines, and in the upper and lower extremities, stroke is the most usual common manifestation of paradoxical embolism. the prevalence of pfo is estimated to be 45% among patients with cryptogenic stroke or transient ischemic attacks. in a recent study the most common reason for the primary referral of patients with pfo - related conditions for pfo closure (n=416) was cryptogenic stroke (n=219). noncerebral paradoxical embolism was rare (n=12) and 75% of noncerebral embolisms were myocardial infarctions (n=8). whereas myocardial infarction is a rare complication of paradoxical embolism through pfo, there are even fewer reports of renal infraction in patients with pfo. most patients reported with paradoxical renal infarction present with multiorgan involvement and have multiple acute ischemic symptoms, such as dyspnea and abdominal pain. this case is unique in that the patient had acute flank pain and the embolism was confirmed only in the kidneys, with no other organ involvement. secondary prophylaxis for a paradoxical embolism can involve pharmacotherapy or the closure of the right - to - left shunt, which can be performed surgically or with a transcatheter procedure. the efficacy of the devices used to prevent recurrent systemic arterial embolisms, and in particular cerebrovascular embolisms, is unknown but is currently under investigation in large - scale clinical trials. closure of the pfo after the first embolism is recommended for patients at high risk of recurrent embolic events. the risk factors associated with embolic recurrence include atrial septal aneurysm, high shunting volume, and shunting at rest. other risk factors include large pfo (more than 3.4 mm), higher mobility of the pfo valve, a well developed eustachian valve, a valsalva maneuver immediately prior to event, and a history of recurrent embolic events. pfo can be closed percutaneously with a low rate of significant residual shunting and very few complications. after closure of the pfo, no patient experienced a recurrent paradoxical embolic event during the medium - term (up to four years) follow - up period. in this case, the patient also had long - standing itp when his renal infarction occurred. his platelet count had not changed for 10 years, without treatment. given that his coagulation profile was normal and there was no thrombosis in the renal artery, because he had no obvious source of emboli, we used contrast echocardiography and a bubble test during the valsalva maneuver to examine the possibility of a paradoxical embolism and confirmed the diagnosis of paradoxical renal infarction caused by pfo. echocardiography shows that he has a shunting not only at valsalva but also at rest. and the patient was suspected of having had recurrent renal infractions in the past based on mr angiography findings. so, secondary prophylaxis was required to prevent additional embolisms. because he suffered hemorrhoidal bleeding during heparin treatment and his platelet count was low, the insertion of a transcatheter pfo closure device was planned, considering the potential problems associated with maintaining oral anticoagulation therapy. in conclusion, the role of pfo as a source of ischemic events in various organs is becoming increasingly evident. this case suggests that renal infarction can be caused by a paradoxical embolus from a pfo. paradoxical embolism is a rare cause of renal infarction, but immediate recognition of a paradoxical embolus is very important so that anticoagulation or device closure can prevent further embolic infarctions in other organs, as well as in the kidneys. if renal infarction occurs repeatedly, with no evident cause of the thromboembolism or during anticoagulation therapy, a paradoxical embolism through a pfo should be considered.	a 48-year - old man presented with acute right flank pain. a computed tomography scan revealed right renal infarction. because he had no thrombosis in the renal vessels and no clear embolic source, a further examination was performed to find the cause of the renal infarction. on transesophageal echocardiography, a right - to - left shunt during the valsalva maneuver established a diagnosis of patent foramen ovale. this is a case of paradoxical embolism through a pfo leading to renal infarction.
the concept of total mesorectal excision (tme) has been the most important development in rectal cancer surgery during the last two decades. after the introduction of tme the rate of local recurrence could be dramatically reduced. even without curative approach, local recurrence was reduced to 6 - 12% and 5-year survival rate improved to 53 - 87% [2 - 4 ]. however, it is noteworthy, that the excellent results of a local recurrence rate of less than 5% without neoadjuvant treatment modalities as reported by heald have not been reached by the majority of rectal surgeons. quality of mesorectal excision according to m.e.r.c.u.r.y. criteria. a recent multicentre trial analysed the benefit of preoperative radiation before tme surgery. if optimal tme - quality could be achieved in a controlled scientific trial in only 50% of patients, serious concern should arise about the tme quality in the absence of pathological quality control. although most centres claim performing tme surgery, the literature evaluating tme quality is scarce. to close this gap we present our results of tme surgery after the introduction of quality controls for rectal cancer surgery at our centre in 2004. during a period of 36 months, between january 2004 and december 2006, 103 patients underwent surgical resection for rectal cancer at the department of surgery, st. josef hospital, ruhr- university bochum, germany. sixty percent (62/103) were male, 40% (41/103) were female. preoperative staging included complete colonoscopy or barium enema, abdominal ct scan and chest x - ray. the decision about neoadjuvant therapy was based on weekly multidisciplinary tumour board reviews. in 29% (30/103) neoadjuvant therapy was performed prior to operation, including 22% (23/103) short term radiation (5 5 gy) and 7% (7/103) long term chemoradiation (50 gy). in - hospital death after anterior resection, reconstruction was achieved via stapled anastomosis (29 mm or 31 mm stapler) or hand sutured coloanal anastomosis. reconstruction included the formation of a colonic pouch by performing a transverse coloplasty whenever possible. the quality of the surgical resections was first judged by macroscopic assessment of the specimens ' surface. after fixation, staining and slicing, the completeness of the mesorectum was judged by microscopic investigation. tumour type, t - stage and n - stage were documented for the purposes of the study. further recording included proximal, distal and circumferential resection margins, tumour size and histopathological grading. after anterior resection, reconstruction was achieved via stapled anastomosis (29 mm or 31 mm stapler) or hand sutured coloanal anastomosis. reconstruction included the formation of a colonic pouch by performing a transverse coloplasty whenever possible. the quality of the surgical resections was first judged by macroscopic assessment of the specimens ' surface. after fixation, staining and slicing, the completeness of the mesorectum was judged by microscopic investigation. tumour type, t - stage and n - stage were documented for the purposes of the study. further recording included proximal, distal and circumferential resection margins, tumour size and histopathological grading. 8% (8/103) of patients needed urgent surgery secondary to ileus (3% ; 3/103) or perforation of rectal carcinoma (5% ; 5/103). in 76% (78/103) an anterior resection was performed, 6% underwent (6/103) hartmann 's operation and 2% (2/103) had a colectomy. in 16% (17/103) of cases these patients either had local recurrence of rectal cancer, a tumour less than 2 cm from the anal verge, sphincter infiltration, sphincter insufficiency or malignant melanoma of the anus. major surgical complications occurred in 17% (18/103) of patients, including anastomotic leakage, wound dehiscence, intra - abdominal abscess formation, postoperative haemorrhage and failure of the rectal remnant after hartmann ' operation. major general complications occurred in 7% (7/103) including pneumonia, stroke and myocardial infarction (table 2). mortality after elective operations was 4% (4/95), while patients undergoing urgent surgery had a mortality rate of 38% (3/8), p = 0.026. an evaluation of the quality of the mesorectum was possible. in 10% (10/103) 7 patients had local recurrence of rectal cancer and had already had rectal resection during prior surgery. 3 patients were part of another study which required immediate opening of the removed rectum for the collection of unfixed tumour biopsies from the removed tumour. i) in 99% (92/93) of removed tumours. in 1% (1/93) ii), and none (0/93) of the removed tumours had an incomplete mesorectum (m.e.r.c.u.r.y. macroscopic high tme quality of an unfixed rectal resection specimen showing the intact mesorectum without defects on the surface. uicc tumour stage ; final histology (n = 103). in 98% (91/93) the circumferential resection margin was negative, showing no tumour infiltration at least 2 millimeters away from the lateral resection margin. one patient requiring urgent surgery for perforated rectal cancer (uicc tumour stage iiic) had involvement of the lateral resection margin. the other patient with lateral resection margin involvement was operated after long term chemoradiation (uicc tumour stage iiib). fifty percent (51/103) of patients were staged to have advanced rectal cancer (> t2 or n+). of those, 61% (31/51) received neoadjuvant radiation, while 39% (20/51) underwent operation without preoperative radiation. the reasons for primary operation without radiation in advanced stages of rectal cancer included recurrence of rectal cancer (7% ; 7/103), suspected metastatic disease (9% ; 9/103), urgent surgery (8% ; 8/103), advanced age (2% ; 2/103), severe co - morbidities (2% ; 2/103), incompliance (1% ; 1/103), other malignancy (1% ; 1/103) or combination of these. the mean number of lymph nodes removed during the operation was 19 (range 4 - 47). sixtysix percent of the removed tumours (68/103) had moderate differentiation, 17% (18/103) good differentiation and 9% (9/103) poor differentiation, while in 1% (1/103) an un differentiated tumour was found. in 7% (7/103) of cases grading was not possible. distant metastases were pre sent in 9% (9/103), including 6 patients with hepatic metastases, 1 patient with pulmonary and hepatic meta stases and two patients with metastases towards other organs. in 91% (94/103) patients had no distant metastases. in 2% (2/103) of the cases a macroscopic residual tumour (r2) was present in the pelvis after resection. 5% (5/103) of the patients had overall r2 resection because of hepatic or pulmonary metastasis, while 2% (2/103) of the patients underwent simultaneous hepatic tumour resection resulting in r0 situation. until may 2009 this patient had palliative rectal resection for a pt3b, pn2 (8/22), l1, v1, pn1, pm1 (hepatic and pulmonary), g3, uicc - stage iv rectal cancer. 8% (8/103) of patients needed urgent surgery secondary to ileus (3% ; 3/103) or perforation of rectal carcinoma (5% ; 5/103). in 76% (78/103) an anterior resection was performed, 6% underwent (6/103) hartmann 's operation and 2% (2/103) had a colectomy. in 16% (17/103) of cases these patients either had local recurrence of rectal cancer, a tumour less than 2 cm from the anal verge, sphincter infiltration, sphincter insufficiency or malignant melanoma of the anus. major surgical complications occurred in 17% (18/103) of patients, including anastomotic leakage, wound dehiscence, intra - abdominal abscess formation, postoperative haemorrhage and failure of the rectal remnant after hartmann ' operation. major general complications occurred in 7% (7/103) including pneumonia, stroke and myocardial infarction (table 2). mortality after elective operations was 4% (4/95), while patients undergoing urgent surgery had a mortality rate of 38% (3/8), p = 0.026. in 90% (93/103) an evaluation of the quality of the mesorectum was possible. in 10% (10/103) 7 patients had local recurrence of rectal cancer and had already had rectal resection during prior surgery. 3 patients were part of another study which required immediate opening of the removed rectum for the collection of unfixed tumour biopsies from the removed tumour. i) in 99% (92/93) of removed tumours. in 1% (1/93) ii), and none (0/93) of the removed tumours had an incomplete mesorectum (m.e.r.c.u.r.y. macroscopic high tme quality of an unfixed rectal resection specimen showing the intact mesorectum without defects on the surface. uicc tumour stage ; final histology (n = 103). in 98% (91/93) the circumferential resection margin was negative, showing no tumour infiltration at least 2 millimeters away from the lateral resection margin. one patient requiring urgent surgery for perforated rectal cancer (uicc tumour stage iiic) had involvement of the lateral resection margin. the other patient with lateral resection margin involvement was operated after long term chemoradiation (uicc tumour stage iiib). fifty percent (51/103) of patients were staged to have advanced rectal cancer (> t2 or n+). of those, 61% (31/51) received neoadjuvant radiation, while 39% (20/51) underwent operation without preoperative radiation. the reasons for primary operation without radiation in advanced stages of rectal cancer included recurrence of rectal cancer (7% ; 7/103), suspected metastatic disease (9% ; 9/103), urgent surgery (8% ; 8/103), advanced age (2% ; 2/103), severe co - morbidities (2% ; 2/103), incompliance (1% ; 1/103), other malignancy (1% ; 1/103) or combination of these. the mean number of lymph nodes removed during the operation was 19 (range 4 - 47). sixtysix percent of the removed tumours (68/103) had moderate differentiation, 17% (18/103) good differentiation and 9% (9/103) poor differentiation, while in 1% (1/103) an un differentiated tumour was found. in 7% (7/103) of cases grading was not possible. distant metastases were pre sent in 9% (9/103), including 6 patients with hepatic metastases, 1 patient with pulmonary and hepatic meta stases and two patients with metastases towards other organs. in 91% (94/103) patients had no distant metastases. in 2% (2/103) of the cases a macroscopic residual tumour (r2) was present in the pelvis after resection. 5% (5/103) of the patients had overall r2 resection because of hepatic or pulmonary metastasis, while 2% (2/103) of the patients underwent simultaneous hepatic tumour resection resulting in r0 situation. until may 2009 this patient had palliative rectal resection for a pt3b, pn2 (8/22), l1, v1, pn1, pm1 (hepatic and pulmonary), g3, uicc - stage iv rectal cancer. the prognosis of rectal cancer depends principally on tumour stage at the time of diagnosis, while local recurrence depends rather on surgical technique. in studies on colorectal cancer, survival is most important, but trials on rectal cancer also focus on local recurrence since local control also correlates with survival. patients receiving preoperative radiotherapy before conventional resection for rectal cancer still have recurrence rates between 28% and 37%. the rate of local recurrence is directly related to the technique of rectal cancer excision, which makes surgical technique the most important factor for patients ' outcome. although tme is accepted in western countries as a standard principle of surgical treatment for rectal cancer the rate of local recurrence without radiotherapy varies between 3% and 18%. kapiteijn and his group could show that short term preoperative radiotherapy reduced the rate of local recurrence. but the fact that best results for rectal cancer surgery were reported without radiotherapy by heald who originally introduced the principle of tme makes it obvious that the surgeon plays a crucial rule for patients ' prognosis. at a mean follow up of 4,2 years after curative resection for rectal cancer heald reported a local recurrence rate of less than 3%. 1. is the variation in local control in studies related to the surgical quality of tme ? although modern surgical treatment of colorectal cancer implies the operative principle of tme, data on tme quality of resected specimen are rare. tme quality was optimal in 47%, 40% of patients had nearly complete tme, while 13% had incomplete tme. the rate of local recurrence was 41% (59% overall recurrence), in patients with nearly complete tme the rate of local recurrence was 6% (17% overall recurrence), while in patients with optimal tme surgery the rate of local recurrence was less than 2% (2% overall recurrence). i), but there were 12 patients with positive resection margins, which makes local recurrence likely to occur. hermanek and heald also focused on the results of the rectal cancer study from the netherlands. they showed that not the whole series of the dutch trial did represent a standardized tme surgery since there was a high rate of incomplete mesorectal excisions for resectable rectal cancer. the variation in local control therefore seems to be an indicator for differences in surgical quality of tme. more evidence for the importance of surgical quality is the difference between multicentre trials and results from single institutions. heald 's excellent data were the result of a single person applying a new technique. the analysis from the netherlands might have compaired excellent surgical quality with poorer quality from different institutions. the role of the surgeon becomes even more obvious since objective analysis of the operative quality is possible by the evaluation of the quality of tme. therefore the documentation of tme quality by pathologists is essential to detect deficits in surgical technique. this may lead to better tme quality and seems to be an effective tool to improve operative results. in our series we could demonstrate that optimal tme quality can be achieved with different operative procedures in any stage of rectal carcinoma. although we had a relatively high rate of abdominoperineal resections this did not increase the rate of incomplete tme. other institutions showed that optimal tme quality can be achieved by individual training after instruction by tme trained surgeons. all operations except for urgent surgery were supervised by surgeons being experienced in tme surgery for several years. during the whole study operations were performed by 7 different surgeons from our institution. the introduction of tme surgery can be associated initially with higher rates of anastomotic leakage, but several studies showed that these findings would improve during the routine application of tme surgery [21 - 23 ]. during the time of our study there were 4 of 78 cases with a clinical apparent anastomotic leakage. an explanation for the high rate of hartmann 's stump leakage is that these were all urgent operations for perforated rectal cancer in patients requiring high dose catecholamines imparing wound healing. two patients died secondary to generalised peritonitis after hartmann 's operation, the other patient died due to excessive liver metastases after urgent colectomy. in our series the mean number of removed lymph nodes was 19 (range 4 - 47). patients with less than 12 lymph nodes removed during the operation either had a local recurrence of rectal cancer or had undergone neoadjuvant radiation. apart from tme quality another marker for mastery in rectal cancer surgery is the achievement of negative resection margins. a distance of more than 1 mm from the tumour to the border of resection is considered to be a negative margin, although a recent analysis reported a distance of 2 mm to be the limit. there may be further discussion about the exact distance for negative resections margins, but the optimal technique to obtain free resection margins is total mesorectal excision since it has been shown that tme achieves a negative resection margin in up to 96% of resected specimen. they analysed the importance of tme to obtain free circumferential resection margins showing that in patients with positive crm the rate of incomplete tme was 44% while in patients with negative crm the rate of incomplete tme was only 11%. furthermore lateral margin involvement was more likely to occur in advanced tumour stages than in tumours with positive lymph nodes. in uicc stage iii with positive crm secondary to incomplete mesorectal excision there were significantly more patients with lateral margin involvement, by the primary tumour than by positive lymph nodes. compared to the results from the literature ranging between 18% and 28% of resection margin involvement our findings reflect another effect of optimal tme surgery. taken both together, the accuracy of mesorectal excision and the analysis of circumferential resection margins are effective in predicting patients ' prognosis. negative resection margins unfortunately do not only depend on surgical technique but on tumour size and tumour stage at the time of operation. although radiotherapy has been shown to improve outcome for patients with resectable rectal cancer, subgroup analysis from the dutch trial showed that there is no benefit for patients with uicc tumour stage i or iv in the upper part of the rectum. therefore it is important to improve selection criteria for the application of preoperative radiotherapy to protect patients from the side effects of radiation without benefit. in our analysis we could show that optimal tme quality is feasible in all stages of rectal cancer. high quality tme surgery can be performed with a rate of morbidity and mortality comparable to the results from the literature. with adequate surgical expertise high quality tme surgery future studies should evaluate outcome and local recurrence in accordance to the degree of tme quality.	backgroundthe concept of total mesorectal excision has revolutionised rectal cancer surgery. tme reduces the rate of local recurrence and tumour associated mortality. however, in clinical trials only 50% of the removed rectal tumours have an optimal tme quality. patients : during a period of 36 months we performed 103 rectal resections. the majority of patients (76% ; 78/103) received an anterior resection. the remaining patients underwent either abdominoperineal resection (16% ; 17/103), hartmann 's procedure (6% ; 6/103) or colectomy (2% ; 2/103).resultsin 90% (93/103) tme quality control could be performed. 99% (92/93) of resected tumours had optimal tme quality. in 1% (1/93) the mesorectum was nearly complete. none of the removed tumours had an incomplete mesorectum. in 98% (91/93) the circumferential resection margin was negative. major surgical complications occurred in 17% (18/103). 5% (4/78) of patients with anterior resection had anastomotic leakage. 17% (17/103) developed wound infections. mortality after elective surgery was 4% (4/95).conclusionoptimal tme quality results can be achieved in all stages of rectal cancer with a rate of morbidity and mortality comparable to the results from the literature. future studies should evaluate outcome and local recurrence in accordance to the degree of tme quality.
a growing body of literature has provided robust evidence that formalin fixed, paraffin - embedded (ffpe) tissue can be successfully analyzed using mass spectrometry - based proteomic methods, enabling the use of these archival specimens for biomarker discovery through retrospective analysis. although qualitative protein identifications can be obtained from fixed tissue, fixation leads to covalent chemical modification and cross - linking of proteins, dna and rna, which may be expected to affect the quantitative reliability of ffpe tissue analyses by targeted proteomic methods, such as multiple reaction monitoring (mrm). recently, research efforts have begun to demonstrate the technical feasibility of targeted quantitative proteomic analysis in ffpe tissue. huang. described the shotgun proteomic analysis of laser capture microdissected ffpe primary and metastatic melanomas and identified 120 proteins as potential markers of metastasis. label - free quantitation was performed with extracted ms1 signal from an ion trap ms instrument. analyzed calcyclin peptides by selected reaction monitoring (one transition per peptide) on a triple quadrupole instrument with sequence analogue peptides as reference standards. similarly, nishimura. quantified two potential prognostic markers for lung adenocarcinoma in ffpe tissue using an endogenous -actin peptide as an internal standard. although these reports have demonstrated the application of targeted protein analyses of ffpe tissue, the critical issue of how formalin fixation affects the precision of quantitative analyses in ffpe tissues remains unexplored. because formaldehyde chemistry significantly affects lysine c - terminal peptides, it is not clear whether quantitative strategies must be adjusted to avoid these. to assess the impact of fixation on the reproducibility of peptide quantitation, we compared the mrm signals for tryptic peptides derived from paired fixed and frozen clear cell renal cell carcinoma (rcc) tissue from the same tumor. our analyses describe the precision of measurements of a large set of tryptic peptides across a broad concentration range in both ffpe and frozen specimens. we further evaluated the precision of mrm - based quantitation through the analysis of peptides from the her2 (erbb2) receptor in ffpe and frozen tissues from a mouse xenograft model of her2 overexpressing breast cancer. finally, we used mrm analyses to quantify her2 protein in ffpe specimens of immunohistochemically confirmed her2-positive and her2-negative human breast cancers. sub - x xylene substitute was obtained from surgipath (richmond, il). iodoacetamide (iam) was from sigma (st. louis, mo), tris - carboxyethylphosphine (tcep) was from pierce (rockford, il), sequencing grade trypsin was from promega (madison, wi), trifluoroethanol and dithiothreitol (dtt) were from acros (geel, belgium). trifluoroacetic acid, ammonium bicarbonate, and urea were purchased from thermofisher scientific (waltham, ma). the deidentified human tissue samples and experimental protocol were subject to institutional review board exempt approval (institutional review board protocols 080856 and 110453). fixed and frozen rcc tissues were obtained from the cooperative human tissue network - western division (vanderbilt university, nashville, tn). ffpe human breast cancer tissue blocks were obtained through the vanderbilt - ingram cancer center breast cancer spore tissue core. fixed and frozen mouse xenografts of bt474 (her2 +) and sum159 (her2 negative) cell lines were kindly provided by the laboratory of carlos l. arteaga. paraffin was removed with three washes of 1 ml of sub - x and tissue was rehydrated with 2 1 ml washes each of 100%, 85%, and 70% ethanol. deparaffinized, rehydrated tissue slices were resuspended in 100 l of ammonium bicarbonate (100 mm, ph 8.0) and were heated at 80 c for 2 h. tryptic digestion was done by an adaptation of the method of wang. trifluoroethanol (tfe) (100 l) was added and the samples were sonicated for 20 s followed by 30 s incubation on ice. the resulting homogenate was heated for 1 h at 60 c followed by a second series of sonication steps, as described above. the homogenate was reduced with tris-(carboxyethyl)phosphine (10 mm) and dithiothreitol (25 mm) at 60 c for 30 min, followed by alkylation with iodoacetamide (50 mm) in the dark at ambient temperature for 20 min. the reduced and alkylated protein mixture was diluted to 1 ml with ammonium bicarbonate (50 mm, ph 8.0) followed by addition of trypsin at 1:50 (w / w). the digest was incubated overnight at 37 c, followed by freezing at 80 c and lyophilization. samples were resuspended in 1 ml of water, desalted over 1 cm (100 mg) sep - pak vac c-18 cartridges (waters corp., milford, ma), and evaporated to dryness in vacuo with a speed - vac sample concentrator (thermofisher, waltham, ma). isoelectric focusing (ief) of tryptic peptides was performed by a modification of the method described previously. rcc tryptic peptides (200 g) were resuspended in 500 l of 6 m urea and loaded in an ipgphor rehydration tray. immobiline immobilized ph gradient strips (24 cm, ph 3.54.5) were placed over the samples and allowed to rehydrate overnight at ambient temperature. the loaded strips were focused at 21 c on an ettan ipgphor-3 ief system (ge healthcare, piscataway, nj) using the following program : step at 300 v for 900 vh ; gradient to 1000 v for 3900 vh ; gradient to 8000 v for 13500 vh ; step to 8000 v for 93 700 vh. the strips were then cut into 20 (1.2 cm) pieces and placed in separate wells of a 96-well elisa plate. peptides were eluted from the strips as follows : 200 l of 0.1% formic acid (fa) for 15 min ; 200 l of 50% acetonitrile (acn)/0.1% fa for 15 min ; 200 l of 100% acn/0.1% fa for 15 min. solutions of extracted peptides were evaporated in vacuo, resuspended in 1 ml 0.1% trifluoroacetic acid, and desalted over a 96-well c18 oasis hlb plate 30 m (10 mg) (waters corp., peptide solutions were evaporated in vacuo, resuspended in 100 l of 0.1% fa, and placed in sample vials for liquid chromatography tandem mass spectrometry (lc ms / ms) analysis. ms / ms analyses were performed on an ltq - xl mass spectrometer (thermo electron, san jose, ca) equipped with an eksigent 1d nanolc and microautosampler (dublin, ca). peptides were loaded on a 100 m 5 cm fused silica capillary guard column (polymicro technologies, llc., phoenix, az) packed with 5 m, 300 jupiter c18 (phenomenex, torrance, ca) and resolved on a 100 m 11 cm fused silica capillary column (polymicro technologies, llc., phoenix, az) packed with 5 m, 300 jupiter c18 (phenomenex, torrance, ca). liquid chromatography was carried out at ambient temperature at a flow rate of 0.6 l min using a gradient mixture of 0.1% (v / v) formic acid in water (solvent a) and 0.1% (v / v) formic acid in acn (solvent b). peptides eluting from the capillary tip were introduced into the ltq source in microelectrospray mode with a capillary voltage of approximately 2 kv. a full scan was obtained for eluting peptides in the range of 4002000 amu followed by six data - dependent ms / ms scans. ms / ms spectra were recorded using dynamic exclusion of previously analyzed precursors for 60 s with a repeat of 1 and a repeat duration of 1. ms / ms spectra were generated by collision induced dissociation of the peptide ions at a normalized collision energy of 35% to generate a series of b- and y - ions as major fragments. mrm analyses were performed on a tsq vantage triple quadurpole mass spectrometer (thermo electron, san jose, ca) equipped with an eksigent ultra nanolc and microautosampler (dublin, ca). ms / ms analyses, except that the flow rate was reduced to 0.4 l min. analyses were performed using the labeled referenced peptide (lrp) method, where peak areas for target peptides were normalized against the isotope labeled -actin peptide standard u c, n - arg - gysftttaer, which was added at a concentration of 25 nm. four transitions were monitored for each peptide and a maximum of 40 peptides were monitored per method. extracted ion chromatogram peak areas were measured as the sum of the peak areas for the four monitored transitions. transitions were selected from the most intense y - ions observed in a spectral library derived from shotgun data sets. the scansifter algorithm read tandem mass spectra stored as centroided peak lists from thermo raw files and transcoded them to mzml files. if 90% of the intensity of a tandem mass spectrum appeared at a lower m / z than that of the precursor ion, a single precursor charge was assumed ; otherwise, the spectrum was processed under both double and triple precursor charge assumptions. tandem mass spectra were assigned to peptides from the ipi human database version 3.37 (may 5, 2008 ; 69 238 sequences) by the myrimatch algorithm, version 1.6.75. the sequence database was doubled to contain each sequence in both normal and reversed orientations, enabling false discovery rate estimation. myrimatch was configured to expect all cysteines to bear carboxamidomethyl modifications and to allow for the possibility of oxidation on methionines. candidate peptides were required to feature trypsin cleavages or protein termini at one end (semitryptic search) ; any number of missed cleavages was permitted. a precursor error of 1.25 m / z was allowed, but fragment ions were required to match within 0.5 m / z. the idpicker algorithm v2.6.165.0 filtered the identifications for each lc ms / ms run to include the largest set for which a 5% identification false discovery rate could be maintained, as described by qian., and applied parsimonious protein assembly, reporting the smallest list of proteins which could account for the identified peptides. proteins were required to have at least two different peptide sequences observed within an ief sample set. database protein entries that could not be distinguished based on the observed peptides were combined into protein groups representing the most parsimonious assignment of the spectral count data. false discovery rates (fdr) rates were computed by the formula : the algorithm reported the number of spectra and number of distinct sequences observed for each protein and protein group in each sample set. shotgun proteome analysis of fixed and frozen rcc tissue indicated a qualitative concordance in protein groups identified in both ffpe and frozen tissue (figure 1a), as we reported previously for comparison of frozen and ffpe tissues. (spectral count data for the identified proteins is presented in supporting information table s1 ; a full summary of the data set is provided in the accompanying idpicker report. of 2165 total protein groups identified, 91% were found in both sample types. however, the resulting peptide identifications obtained from tryptic digests of fixed tissue were biased against lysine c - terminal peptides (figure 1b). whereas in frozen tissue the ratio of lysine c - terminal peptides to arginine c - terminal peptides was 1.11, the ratio in ffpe samples was reduced to 0.93. these observations are in agreement with our previous analyses of frozen and ffpe colon adenocarcinoma tissue and are consistent with the known reactivity of aldehydes toward primary amines, which preferentially consumes lysine residues and thus affects the yield of lysine c - terminal peptides. we thus asked whether the modification of lysine residues would affect the quantitation of lysine c - terminal peptides in a tryptic digest of ffpe tissue. high qualitative concordance was observed for proteins identified in both ffpe and frozen tissue digests. peptides observed in ffpe tissue were biased against lysine c - terminal peptides, indicated by a lower lysine to arginine peptide ratio. error bars represent standard deviation from 4 ief replicates. to assess measurement precision in ffpe tissue, 114 peptides were selected as mrm targets from a list of peptides identified from shotgun proteomic analysis of ief - fractionated tryptic digests of ffpe and frozen rcc tissue (supporting information table s2). the peptides selected spanned the entire range of peptide and protein abundance, as determined by spectral count information in the shotgun data set (supporting information table s1). the 114 candidates each were between 7 and 25 amino acids in length ; contained no methionine, cysteine, histidine, n - terminal glutamine residues or missed tryptic cleavages ; were unique to a single protein in the search database ; and were observed predominantly as doubly charged precursors. transitions were chosen from the 4 most intense y - ions based on reference spectra from the shotgun data set. the precision with which a peptide can be measured and the intensity of the signal obtained are a function of the peptide concentration, ionization and fragmentation characteristics, variability in yields of sample processing steps (e.g., digestion), and the characteristics of the instrument used to measure the analyte. the measurement precision of the instrument can be empirically assessed through the analysis of an internal standard of known concentration and can provide a reference for the measurement of effects on precision caused by the fixation process. thus, in addition to the 114 candidate peptides, an endogenous peptide of sequence gysftttaer, corresponding to -actin, was monitored, as well as an isotope labeled version of the gysftttaer peptide spiked - in at a constant concentration of 25 nm to provide for signal normalization for the targeted peptides and to monitor measurement precision. five serial sections of ffpe and frozen rcc tissue were processed in parallel and analyzed using 3 mrm acquisition methods, which monitored 40 peptides and 160 transitions each. the methods analyzed groups of peptide targets corresponding to proteins of different abundances, based on spectral counts from the shotgun analyses. median spectral counts for the proteins covered in methods 1, 2, and 3 were 190, 129, and 38, respectively. the proteins and peptides comprising the methods are listed in supporting information table s3. lists of the transitions monitored for each peptide are provided as skyline (.sky) files in the supporting information. the coefficient of variance (cv) for the lrp - normalized peak area over triplicate mrm runs was determined for each peptide. given the possible effects of formaldehyde fixation chemistry on lysine c - terminal tryptic peptides, the precision of mrm measurement was analyzed separately for lysine and arginine c - terminal peptides. the median cvs for the 52 lysine c - terminal peptides were not significantly different between analyses of fixed (median cv = 0.195) or frozen tissue (median cv = 0.190) using the mann median cvs for analyses of the 58 arginine c - terminal peptides also indicated no significant difference between fixed (median cv = 0.183) or frozen tissue (median cv = 0.180, figure 2). this result suggests that there is no discernible effect of formaldehyde fixation chemistry that affects the reproducibility of peptide mrm measurement. in addition, there was no significant difference in median cv between lysine c - terminal peptides (median cv = 0.195) and arginine c - terminal peptides (median cv = 0.183) from ffpe tissue, suggesting that mrm measurements of ffpe tryptic peptides display similar technical variation, regardless of the c - terminal amino acid. cvs for mrm measurements of 52 lysine c - terminal peptides and 58 arginine c - terminal peptides in 5 serial sections of frozen and ffpe rcc samples. comparison of median cvs for peptide measurements from ffpe and frozen tissues indicated no significant effect of fixation on the precision of peptide measurement (mann whitney u test). the influence of fixation status on individual peptides was further assessed by considering the median cv obtained for analyses of each peptide across the 5 serial slices (supporting information table s4). a commonly used method to establish the limit of quantitation (loq) for an analyte is to determine the average signal at the retention time of interest in a blank sample plus 8 standard deviations of the average blank signal. for mrm analyses, signals from a blank run at a given peptide m / z value and retention time can result in very small values, which can yield theoretical loqs far below the practical limitations of the analytical system. a more conservative loq estimate can be obtained empirically by determining the concentration at which the cv of replicate measurements exceeds 25%. of the 114 peptides analyzed, 10 were deemed to be undetectable due to lack of a consistent signal among replicates and samples. of the remaining 104 peptides, 37 yielded median cvs greater than 25% in the ffpe derived samples. these peptides were thus designated as being below the loq in these samples. in analyses of frozen tissue digests, 27 peptides exceeded the 25% median cv threshold (supporting information table s3). a greater number of peptides below the loq in ffpe tissues may reflect reduced signal intensity, most likely due to reduced tryptic peptide yield in digests of ffpe samples. in total, 46 peptides yielded median cvs in excess of 25% in either ffpe or frozen tissue ; 18 of these had median cvs greater than 25% in both tissue types. overall, 51% of the peptides targeted (58/114) yielded signals estimated to be above the loq. this success rate for analyses in ffpe tissues is comparable to other empirical assessments of proteotypic peptide suitability in semiquantitative mrm assay development for biomarker verification. as is evident in supporting information table s3, methods 2 and 3 produced increasing numbers of peptides exceeding the 25% median cv threshold and this effect was somewhat greater for ffpe tissue. peptides exceeding the threshold were as follows : method 2, 16 peptides in ffpe, 9 in frozen ; method 3, 21 in ffpe, 18 in frozen. peptides targeted by these two methods were from proteins represented with fewer spectral counts in the shotgun analysis (supporting information table s2), indicating lower abundance in the rcc tissues. the most likely explanation for the effect on median cv in the ffpe samples is the presence of protein cross - links, which result in a lower yield of proteotypic tryptic peptides detectable by mrm analysis, thus decreasing concentrations of the peptides below the limit of quantitation. the influence of fixation appears not to be specific to a particular class of protein, or subcellular compartment, but rather is distributed uniformly among proteins we analyzed. in mrm analyses, effects on peptide yield will have the greatest impact on quantification of peptides that are already near the limit of quantitation in unfixed, frozen samples. this interpretation is consistent with our observation here (figure 1 and supporting information table 1) and previously that the proteins represented in shotgun data sets with fewer spectral counts yield fewer peptide and protein group identifications in ffpe tissue than in frozen specimens in shotgun proteomic analysis of ief - fractionated samples. we also considered the effect of fixation on the magnitude of the peak areas observed during mrm analysis, a more direct measurement of relative peptide abundance. of the 114 peptides monitored, 70% showed a significant difference in average normalized peak area between fixed and frozen samples (two tailed t test, p < 0.05, supporting information figure 1). figure 3 plots the log2 ratio of lrp - normalized peak areas for peptides from ffpe to frozen tissues. these data demonstrate that peptides from ffpe tissue generally yield lower normalized average peak areas than peptides from frozen tissue, given the predominance of log2 (ffpe : frozen) peptide intensity values below zero in figure 3. this result further illustrates the significant impact of fixation on the quantitation of proteins in tissue. peptides are ordered on the x - axis in order of decreasing log2 ratios (y - axis values). log2 ratios less than zero indicate higher peak areas from peptides derived from frozen tissue. a potentially useful application of mrm quantitative analyses in ffpe tissue is the measurement of specific biomarker proteins of clinical interest. this is normally achieved by immunohistochemistry (ihc), which can be limited by assay variability, antibody sensitivity, and specificity. in addition, ihc methods are not quantitative and assessment of ihc is subjective. mrm analyses provide quantitative measurements with the potential for improved specificity, thus overcoming key performance limitations of ihc. to explore the possible clinical application of mrm in ffpe tissue amplification of her2 is observed in 20% of breast cancer cases and patients harboring her2-overexpressing tumors are candidates for her2-targeted therapies. to monitor multiple peptides unique to her2, the peptide exclusion criteria were relaxed allowing for the inclusion of peptides containing cysteine, methionine, and histidines. peptides were still required to be fully tryptic, between 7 and 25 amino acids in length, unique to her2 and to be represented in a reference spectral library. twenty - five peptides met these criteria and were targeted by mrm in a tryptic digest of the her2 overexpressing bt474 human breast cancer cell line. five transitions were monitored per peptide, as selected from the most intense y - ions observed in a reference spectral library. on the basis of this initial screen, 4 peptides were selected that gave the greatest signal intensity and a cv of less than 25% when analyzing 1 g of tryptic digest. to enable quantitation by stable isotope dilution, labeled peptides corresponding to these 4 sequences were obtained and the limit of quantitation was determined in the absence of biological matrix. limit of quantitation was defined as the concentration of peptide where the cv for triplicate measurements exceeded 25%. the peptides dppfcvar and elvsefsr provided the greatest sensitivity, yielding limits of quantitation within a biologically relevant range of 0.33 and 0.1 fmol on - column, respectively (supporting information figure 2). these limits correspond to a lower limit of detection of 12 000 her2 receptors per cell, assuming 200 pg protein per cell and 1 g tryptic digest on - column (i.e., 5000 cell equivalents injected for mrm analysis). detection limits for peptides gqecveecr and npqlcyqdtilwk exceeded 1 fmol on - column and were not considered for quantitation. we then analyzed ffpe and frozen xenograft tissue samples from human bt474 and sum159 (her2-negative) breast cancer cell lines. whereas signals from her2 peptides were readily discernible in bt474 xenografts, her2 peptide signals were absent in sum159 xenografts, consistent with their low level of her2 expression (figure 4). estimates of her2 receptor numbers from quantification of the dppfcvar peptide were 60% higher than from quantification of the elvsefsr peptide. the dppfcvar peptide is derived from the ectodomain of the her2 receptor, whereas elvsefsr is in the intracellular activation domain of the receptor. indeed, bt474 xenografts have been reported to shed the her2 extracellular domain into the circulation. mrm chromatograms for the her2 intracellular domain peptide elvsefsr from analyses of ffpe xenograft specimens derived from the her2-overexpressing cell line bt474 (left) and the non - her2-expressing cell line sum159 (right).. monitored mrm transitions are depicted for the unlabeled, endogenous peptide, and the isotope - labeled internal standards (insets). the intensity scales (y - axis) are identical in the two sets of plots. to explore the application of mrm for analyses of her2 in ffpe human breast tumor tissues, we analyzed 5 her2-overexpressing breast cancer tissues and 5 her2-negative breast cancers, which had been classified based on previous ihc analysis in the vanderbilt clinical immunohistochemistry laboratory. mrm analyses of these specimens (figure 5b) demonstrate a clear difference in signal intensity for her2 peptides between the her2 + and her2 negative tumors and indicate a wide range of biological variability in receptor expression levels. quantification of receptor levels was performed by stable isotope dilution. assuming a yield of 200 pg protein from an average cell, we estimated her2 receptor levels ranging from 110 000 to 468 000 receptors per cell in the her2 positive cancers and fewer than 14 000 receptors per cell in the her2 negative cancers. because of the higher limit of quantification for the dppfcvar peptide, it was not possible to use this peptide to quantify receptor levels in all specimens. in tissues most highly overexpressing her2, quantification of receptor levels based on dppfcvar yielded the same result as elvsefsr quantification, suggesting no evidence of receptor ectodomain shedding in the tissues analyzed. (a) quantification of her2 receptor protein in frozen (red bars) and ffpe (blue bars) bt474 xenograft tissues. her2 protein was quantified based on mrm of peptides representing the extracellular (dppfcvar) and intracellular (elvsefsr) domains of the receptor. plotted values are mean sd for 3 process replicates of one frozen and one ffpe tumor. (b) quantification of her2 receptor protein in 5 human her2-positive and 5 her2 negative human ffpe breast tumor tissues. mrm quantitation was by stable isotope dilution analysis of peptides representing the extracellular (dppfcvar) and intracellular (elvsefsr) domains of her2. the goal of this work was to assess the performance characteristics of protein quantitation by mrm in ffpe tissue and to apply mrm to analyze a clinically relevant biomarker in human ffpe tissue specimens. our studies addressed the hypothesis that protein cross - linking in ffpe tissues would add to variability in mrm measurements, particularly for lysine c - terminal peptides. although peptide yields, particularly for lysine c - terminal tryptic peptides, are lower in ffpe tissues than in frozen tissues, measurement variation is not significantly different between these specimen types. median cvs for analyses of tryptic peptides in ffpe and frozen tissues were all below 20% and were not significantly different between specimen type or between lysine- and arginine c - terminal peptides. the data not only demonstrate that mrm analyses can be performed with equal precision on ffpe and frozen tissues, but also indicate that lysine c - terminal peptides need not be excluded as mrm candidates when working with ffpe tissue. our analyses of her2 protein in ffpe breast tumor xenografts and in human ffpe breast tissue specimens illustrated the feasibility of applying mrm to quantify clinically important tissue biomarkers. the major question regarding the use of proteomic methods to analyze ffpe tissue is the impact of protein cross - linking that occurs during formalin fixation. the terms antigen retrieval and crosslink reversal have been applied to describe the various protocols to prepare ffpe tissue for ihc analysis. although these approaches facilitate ihc, there is no molecular or chemical evidence to demonstrate that formaldehyde - derived cross - links have been reversed. indeed, given the chemical stability of many formaldehyde - derived covalent cross - links in proteins, it is unlikely that any method or reagent could quantitatively reverse cross - links while sparing peptide bonds and other functional groups. our results suggest the chemical modifications induced by formalin fixation decrease the sensitivity of mrm measurements. thus, somewhat fewer targets are accessible to mrm when ffpe tissue is analyzed. the fractional loss of peptide signal is negligible for high - abundance proteins, but proteins near the limit of quantitation in frozen tissue are less likely to be accurately quantified by mrm in ffpe tissue. this limitation may be overcome to some extent through increased sample input and possibly through the use of larger capacity chromatography columns, permitting injection of larger amounts of tryptic digest. however, our data indicate that the overall impact of formalin fixation on peptide yield and signals was significant, yet modest and does not preclude application of mrm to analyze proteins in ffpe tissues. we should also point out that, despite the validity of this general conclusion, certain peptides may be unusually sensitive to formalin fixation and display high measurement variation in ffpe samples. thus, mrm assay development should consider multiple peptides during assay development and optimization. we were unable to assess the accuracy of quantitation in ffpe tissues because the concentrations of the proteins analyzed were not known. spike - in experiments with known amounts of target proteins to assess quantitative accuracy are not possible with ffpe tissues, as the spiked proteins would not control for the formalin fixation steps, which probably have the greatest effect on protein yield. assessment of accuracy in mrm analyses of any sample type is complicated by uncontrolled variability in protein digestion and recovery. however, the goal of mrm analyses usually is comparison of protein levels, rather than assessment of absolute amounts and the underlying assumption is that deviations from accuracy are evenly distributed across all measurements. precise and reproducible measurement of relative differences between sample classes can enable biomarker verification and validation studies. to evaluate mrm analyses of ffpe tissues to quantify a clinically relevant protein biomarker, we analyzed her2 receptor expression levels in her2 positive and her2 negative breast tumors. her2 is an important diagnostic and predictive factor, as patients with her2-positive tumors are candidates for anti - her2 therapies. her2-positive tumors are defined by intense membrane staining in the majority of tumor cells (3 + by ihc) using antibodies directed against the her2 c - terminus or by expression of 2.2 copies of the her2 gene, as determined by fluorescent in situ hybridization (fish). ihc staining or her2 copy number determination in ffpe tissue sections is semiquantitative in nature and results are dependent on antibody performance and operator experience. further, the rate of discordance between ihc and fish can approach 20%, which is significantly below the maximum rate of 5% recommended by the american society of clinical oncologists. an mrm - based approach to analysis of tissue biomarker proteins offers potential advantages over ihc. most significantly, mrm would allow a systematic approach to configuration of targeted assays for tissue proteins, even when antibody reagents validated for ihc are unavailable. this would remove a major barrier to analysis of protein biomarkers in ffpe specimens. mrm analyses also provide additional molecular detail that may not be accessible through ihc. for example, analyses of her2 protein with mrm quantitation of both extracellular and intracellular sequences can extend the specificity of molecular characterization of her2-positive breast cancers. a potential mechanism of resistance to the her2-targeted drug trastuzumab is shedding of the receptor ectodomain, as her2 is subject to proteolytic processing at the plasma membrane by an adam or mmp. her2 measurement based on mrm analysis of intracellular and extracellular peptide sequences provides a more informative assessment of the status of the her2 target than does ihc and fish. mrm could further enable analysis of modified or variant protein forms, which may be difficult to detect selectively with antibody reagents. our analyses of her2 in ffpe tissue specimens illustrate the potential utility of mrm for analysis of a clinically relevant tissue protein biomarker. the data demonstrate that mrm analyses are concordant with her2 status as measured by ihc. an assessment of the performance of mrm for her2 or other tissue biomarkers is beyond the scope of this preliminary study. nevertheless, our results demonstrate that mrm analyses in ffpe and frozen tissues display similar performance characteristics and that measurement precision in the two tissue types is essentially identical. although target peptide yields are lower in ffpe tissues, this has a modest effect on assay sensitivity. our studies were limited to measurements of unmodified peptides and may not be applicable to post - translationally modified (e.g., phosphorylated, n - acetylated) sequences, where the stability of labile modifications during tissue harvest and fixation may affect the results. our findings indicate that effects of formalin fixation on tissue proteins does not present major potential barrier to the development of mrm - based tissue assays for protein expression for research purposes. the data also suggest that further development of mrm instrumentation and approaches may provide powerful new diagnostic tools for the surgical pathology laboratory.	we compared the reproducibility of multiple reaction monitoring (mrm) mass spectrometry - based peptide quantitation in tryptic digests from formalin - fixed, paraffin - embedded (ffpe) and frozen clear cell renal cell carcinoma tissues. the analyses targeted a candidate set of 114 peptides previously identified in shotgun proteomic analyses, of which 104 were detectable in ffpe and frozen tissue. although signal intensities for mrm of peptides from ffpe tissue were on average 66% of those in frozen tissue, median coefficients of variation (cv) for measurements in ffpe and frozen tissues were nearly identical (1820%). measurements of lysine c - terminal peptides and arginine c - terminal peptides from ffpe tissue were similarly reproducible (19.5% and 18.3% median cv, respectively). we further evaluated the precision of mrm - based quantitation by analysis of peptides from the her2 receptor in ffpe and frozen tissues from a her2 overexpressing mouse xenograft model of breast cancer and in human ffpe breast cancer specimens. we obtained equivalent mrm measurements of her2 receptor levels in ffpe and frozen mouse xenografts derived from her2-overexpressing bt474 cells and her2-negative sum159 cells. mrm analyses of 5 her2-positive and 5 her - negative human ffpe breast tumors confirmed the results of immunohistochemical analyses, thus demonstrating the feasibility of her2 protein quantification in ffpe tissue specimens. the data demonstrate that mrm analyses can be performed with equal precision on ffpe and frozen tissues and that lysine - containing peptides can be selected for quantitative comparisons, despite the greater impact of formalin fixation on lysine residues. the data further illustrate the feasibility of applying mrm to quantify clinically important tissue biomarkers in ffpe specimens.
, calcium is a regulator of intracellular processes, activates proteases, and is essential for photosystem ii (psii) maturation and catalytic activity. in industrial processes, calcium oxide plays a role as a heterogeneous catalyst in transesterification reactions, offering the promise of environmentally sustainable exploitation of biofuels. calcium also plays an important role as a promoter in transition metal - mediated heterogeneous processes. additionally, the use of calcium compounds in homogeneous catalysis has received increasing attention, with applications in polymerization catalysis, hydroamination, and hydrosilylation. the earth abundance of calcium, as well as its biocompatibility, provides motivation for further development of ca - based catalysts. in order to understand the transformations that occur at the calcium site, in both chemical and biological processes, one would like to selectively probe the calcium coordination environment. in this sense, ca k - edge x - ray absorption spectroscopy (xas) is an ideal tool. ca k - edge xas results from the excitation of ca 1s electrons to empty molecular orbitals localized on the calcium atom. as such, ca xas should provide a sensitive probe of the ca coordination environment. however, to our knowledge, a thorough investigation of ca k - edge xas has not yet been made. previous ca k - edge xas studies have generally used a fingerprinting approach, and only limited studies exist in which ca k - edge data have been correlated to theory. the majority of the quantitative information that has been obtained from ca xas data has largely relied on the extraction of metal however, due to the large inherent error in establishing coordination numbers from exafs (25%), the ability to determine accurate coordination numbers from exafs alone is limited. however, previous studies have successfully coupled k - edge analysis with exafs analysis to describe previously uncharacterized metal centers in complex systems such as proteins. therefore, the development of ca k - edge xas should significantly aid in the characterization of ca centers. in principle, the information content of ca xas may be greatly enhanced through a quantitative analysis of the pre - edge spectral region, in a manner analogous to previous studies on first row transition metals. herein, we systematically investigate the information content of ca k - pre - edge xas and extend these studies to complex heterometallic mnca clusters with relevance to the oxygen - evolving complex (oec) of psii, which consists of a mn4o5ca cluster with a distorted chair ca k - edge xas data were obtained for a series of six, seven, and eight coordinate calcium compounds (table 1 and figure 1), including molecular and inorganic lattice species, in order to establish the experimental changes that occur in the ca pre - edge xas region upon altering the coordination environment of the ca ion. experimental intensity and energy correlations further, we apply a td - dft protocol, which allows for the calculation of xas pre - edges for both molecular and inorganic lattice systems, with excellent reliability. (7 and 7) and cubane - like complexes (7, 7, 7) are shown. in the 7 complex, a tetrahydrofuran ligand replaces the dimethylformamide seen in the 7 series. having established the correlation between experiment and theory, we then further explore the applicability of this approach to understanding the changes that occur at the ca center in complex heterometallic systems. in particular, structural information is extracted from the changes in the ca k - pre - edge associated with a one electron reduction of the cubane mn3o4ca cluster (figure 1), which serves as a structural reference point of the mn4o5ca cluster in the oec. as calcium is often suggested to play a mechanistic role in water oxidation, the changes that occur at the ca site during the catalytic cycle are of fundamental interest. our results show that direct correlation between the calcium environment and the observed ca k - pre - edge can be made. further, it is demonstrated that the ca site can serve as a reporter for the changes that occur at the mn sites. ca k - edge xas data was collected on 13 model compounds consisting of both molecular and inorganic infinite lattices. a td - dft protocol was developed and correlated to the experimental data to help characterize the ca k - pre - edge. cao (6), caco3 (6), and ca(oh)2 (6) were purchased at the highest available purity and used without further purification. the ligand precursor h3l, where l is a 1,3,5-triarylbenzene - based ligand, was prepared according to reported procedures. the six coordinate { [lmnmn2o(oac)3]2ca } (6) and seven coordinate molecular complexes lmn3cao4(oac)3(thf) (7), lmn3cao4(oac)3(dmf) (7), [lmnmn2cao2(oac)2(dme)(otf)](otf)2 (7), and [lmn3cao2(oac)2(dme)(otf)](otf) (7 ; figure 1) were synthesized as previously described. xas samples of [lmn2mncao4(oac)3(dmf) ] (7) were prepared by the addition of 1 equiv of cobaltocene to a solution of 7 in dmf. the reaction mixture was then transferred to 40 l xas liquid sample holders and frozen within 5 min of reductant addition. attempts to isolate 7 as a solid in analytically pure form have been unsuccessful to date. the ligand precursors h3tpaa (6,6,6-nitrilotris(methylene)tripicolinic acid) and h2dpaea (n, n - bis[6-carboxypyridin-2-yl)methyl]ethylamine) and compound [ca(dpaea)(h2o)(meoh)]2 (8) were prepared according to reported procedures. all other starting materials including the h2bzida ligand (n - benzyliminodiacetic acid) were commercially available. the elemental analyses were carried out with a c, h, n analyzer (sca, cnrs). an aqueous solution of koh (0.1 m) was added to a suspension of h3tpaa (40.0 mg, 94.70 mol) in water (5 ml) until ph 5 was reached, yielding a colorless solution. solid cacl22h2o (14.0 mg, 95.23 mol) was added to the resulting colorless solution, and after a few minutes of stirring, a white precipitate was formed. after stirring the mixture for 15 min, precipitation was completed by cooling the mixture to 4 c for 12 h. the white powder was filtered off, washed with h2o (2 ml), dried under a vacuum, and collected (yield, 20.0 mg, 37%). calcd for c21h18can4o7h2okcl (571.03) : c, 44.17 ; h, 3.53 ; n, 9.81. found : c, 44.32 ; h, 3.50 ; n, 9.68. this can be cocrystallized with the reported trinuclear compound [{ ca(tpaa)(oh2)}2{ca(oh2)4 } ] by slow evaporation of an aqueous solution of tpaa (ph 8), in the presence of 1 equiv of cacl22h2o. an aqueous solution of koh (0.1 m) was added to an h3tpaa (40.0 mg, 94.70 mol) suspension in water (10 ml) until ph 8 was reached, yielding a colorless solution. solid mncl2 (13.3 mg, 105.7 mol) and cacl22h2o (10.0 mg, 68.0 mol) were successively added. x - ray suitable colorless single crystals of [mn2(tpaa)2ca2(oh2)12][mn(tpaa)]225h2o were obtained upon standing for 24 h. these were filtered, washed with cold water (1 ml), and air - dried for a few days (yield, 29.0 mg, 50%). calcd for c84h84n16o36mn4ca213h2o (2427.76) : c, 41.56 ; h, 4.57 ; n, 9.23. found : c, 41.58 ; h, 4.29 ; n, 9.18. an aqueous solution of koh (0.1 m) was added to a suspension of h2bzida (62.0 mg, 277.7 mol) in water (5 ml) until ph 9 was reached, yielding a colorless solution. solid ca(o3scf3)2 (97.0 mg, 286.8 mol) was added, and the resulting colorless solution was stirred for 10 min. x - ray suitable colorless single crystals of 8 were obtained by slow evaporation of the solvent at 20 c. these were filtered, washed with cold water (four drops), and dried on standing for a few days (yield, 33.0 mg, 36%). calcd for c22h34ca2n2o140.1c11h13no4 (652.99) : c, 42.49 ; h, 5.45 ; n, 4.50. single - crystal diffraction data were taken using an oxford - diffraction xcalibur s kappa geometry diffractometer (mo k radiation, graphite monochromator, 0.71073). an absorption correction was applied, using the abspack oxford - diffraction program with transmission factors in the 0.6740.898 range. the molecular structure was solved by direct methods and refined on f by full matrix least - squares techniques using the shelxtl package. all non - hydrogen atoms were refined anisotropically, and hydrogen atoms were found by fourier transformation and refined with individual isotropic displacement parameters. ccdc 995926, ccdc 995927, and ccdc 995923 contain the supplementary crystallographic data for 8, 8, and 8, respectively. a summary of x - ray data collection and structure refinement for 8 is reported in supporting information table s1.1. the x - ray structure of 8, together with the cif file, is provided as supporting information. solid samples were diluted in boron nitride and mounted on ca - free polycarbonate holders. x - ray absorption spectroscopy (xas) data on the seven coordinate calcium species (7, 7, 7, 7, 7) and on the six coordinate calcium complex 6 were measured at the stanford synchrotron radiation lightsource (3-gev ring) beamline 43 equipped with a si(111) double - crystal monochromator. a four - element silicon drift detector (vortex - me4, hitachi) was used to collect the data as fluorescence excitation spectra. samples were maintained at a temperature of 30 k with a liquid he cryostream (oxford) to minimize radiation damage. ca k - edge data were monitored throughout the course of data collection to ensure that no x - ray induced damage occurred. further, to test for nonlocal damage, the mn k - edge was monitored before and after collection of ca xas data on each sample. all other data were collected at beamline a1 (doris light source at desy, 4.5 gev ring) equipped with a si(111) double crystal monochromator and a vacuum sample chamber. solid samples were diluted in boron nitride and finely dispersed on kapton tape (k104 psa polyimide film with silicone adhesive). spectra were calibrated to the maxima of the rising edge feature in calcium acetate monohydrate set at 4050.0 ev. the athena software program, using the autobk algorithm, was used for data reduction and normalization. a linear pre - edge function and a quadratic polynomial for the postedge were used for background subtraction, followed by normalization of the edge jump. in order to extract intensities and energy positions of pre - edge features, lorentzian sum peaks and a cumulative gaussian lorentzian function for the edge jump. all dft calculations were carried out using the orca program. in general, for molecular complexes, crystal structures with the full ligands were used to generate the starting coordinates for geometry optimization calculations. sham method using the tpss functional and a def2-tzvp basis set with a def2-tzvp / j auxiliary basis set, as well as a dft - d3bj dispersion correction. a dense integration grid (orca grid4 = lebedev 302 points) was used for all the atoms except ca and mn where a grid6 (lebedev 590 points) was applied. the geometry - optimized structures were later used for the ca k - pre - edge xas spectra calculations. the 8 polymer was truncated to a [ca2(bzida)2(oh2)6 ] repeating unit, before geometry optimization, as described in supporting information s2. additionally, the model for the 7 complex was based on the crystal structure of the previously published sc analog. the sc atom was replaced with ca, and the structure was optimized, while maintaining mn - ligand bond distances of the original complex. lastly, the model for the xas spectral calculation of 6 was truncated after geometry optimization to encompass the ca center and acetate ligands, with proton capped oxygens instead of mn o bonds (supporting information s2). the truncation of the model was needed due to the large size of the molecular complex. in models for cao (6), caco3 (6), and ca(oh)2 (6), inorganic lattices were built using the effective core potential embedding approach. in the most popular variant of this approach, the quantum cluster (qc) is embedded in an extended point charge field (pc). furthermore, in order to avoid electron attraction or electron flow from the quantum cluster region toward the positive charges at the pc region, a third boundary region (br) is introduced between qc and pc, which is constructed from repulsive capped effective core potentials (c - ecps). in all the cases, the qc is constructed around the central calcium center spanning a 5 radius region around it. the respective atom centers were exchanged by capped effective core potentials (with ecp basis sets sd(10,mwb) for ca and sd(2,mwb) for o and c). finally, the pc region follows the br, extending 15 from the central calcium. the charges for the three regions were distributed such that the net charge for the model is zero and the total charges for the border and point charge region taken together offset the charge on the quantum cluster region. calcium k - edge xas spectra were calculated with a td - dft approach employing the tamm spin unrestricted calculations were carried out using a large integration grid (grid5 = lebedev 434 points) and the bhlyp functional. as previous studies have shown, the choice of functional can impact the calculated xas spectra, we also carried out a detailed study on the functional dependence. the bhlyp functional was found to offer the best results in terms of resolving the pre - edge consistent with previously reported studies (supporting information s3). the rijcosx approximation was implemented to help reduce the computational cost of the td - dft calculations. for these calculations, the def2-tzvp basis set with a def2-qzvpp / jk auxiliary basis set was used on all atoms except calcium where a def2-qzvp basis set was employed. in order to ensure saturation of the particle / hole transitions spanning the pre - edge and near edge region of the ca k - edge spectra, moreover the calculated intensities include electric dipole, magnetic dipole, and quadrupole contributions. ca k - edge data were obtained for a series of 13 inorganic lattice and molecular complexes (figure 1 and table 1). the six coordinate complexes consist of pseudo - octahedral species with increasing complexity of the oxygen - based ligands. cao (6) can be considered octahedral, followed by 6, which has only small distortions from octahedral symmetry and caco3 (6), which has local d4h symmetry. four seven - coordinate complexes, which mimic the mn4o5ca cluster motifs in the oec, were investigated. these complexes can be grouped as having a cubane - like structure (7, 7) and a noncubane - like structure (7) depending on the arrangement of the metal - oxo atoms. furthermore, one - electron oxidized and reduced pairs of these complexes were investigated. the cubane - like complexes can be best described as having c3v symmetry at the calcium center, while the noncubanes favor a c2v symmetry. to offer a full range of accessible coordination geometries, eight coordinate calcium species (8, 8, 8, and 8) having a mixture of amine, carboxylate, and water ligands were also investigated. calcium geometries for 8 and 8 are best described as trigonal dodecahedron (d2d), while 8 and 8 are more pseudosquare antiprismatic having a symmetry best characterized as intermediary between d4d and d2d. the symmetries around the ca centers are approximated to the first coordination sphere ligand atoms. figure 2 shows the ca k - edge spectra for all investigated compounds. in the case of molecular complexes, the rising edges are similar, and the largest variations occur in the pre - edge region. however, in the inorganic infinite lattice series, the rising edge is dominated by a convolution of multiple scattering and absorption processes resulting in significant variability. such features have previously been reproduced using multiple scattering theory ; however this approach fails to capture the pre - edge region, which is an important probe of coordination environment at the absorbing center. for these reasons, we focus on the pre - edge region between 4038 ev and 4042 ev (figure 2 ; right). the low intensities of the features compared to the rising edge are reminiscent of the 1s 3d dipole forbidden transitions of pre - edges in transition metals (tm) and have previously been assigned as such based largely on empirical considerations and, more recently, based on a density of states analysis of cas infinite lattices. x - ray absorption spectra at the ca k - edge of six (bottom), seven (middle), and eight (top) coordinate ca species : full rising edge of the spectra (left) and the pre - edge region (right). six coordinate species are distinguished from seven and eight coordinate centers by the presence of two pre - edge features split by 1 ev and a lower intensity pre - edge. the pre - edge maxima for six coordinate centers occur at 4039.5 ev and 4040.6 ev, thus spanning the energies of the maxima for seven and eight coordinate complexes (figures 2 and 3). structures at 4039.75 ev. furthermore, a one electron chemical reduction of the 7 cubane - like complex [mn3cao4 ] to yield 7 [mn2mncao4 ] results in a 16 3% lower intensity pre - edge. this is not observed upon one electron chemical reduction of the noncubane 7 to yield 7, both of which have pre - edges centered around 4040.17 ev, similar in intensity and energy to eight coordinate calcium complexes. best fit line (red), 95% confidence interval (dashed gray), 95% prediction band (dotted gray). as k - edge xas transition intensities are predominantly dipole dependent (1s np), the variation in pre - edge intensities can in part be rationalized using symmetry arguments. six coordinate pseudo - octahedral complexes are centro - symmetric with little or no p d mixing, resulting in low intensity dipole forbidden (but quadrupole) 1s 3d transitions into the t2 g and eg orbitals. in the seven and eight coordinate complexes under study, this centro - symmetry is broken, p d mixing becomes favored, and an increase in intensity occurs due to an increase in p - character mixing in the d manifold. the pre - edges are most intense for the cubane - like structures because they have pseudo c3v symmetry and p mixing is symmetry allowed with all five d orbitals. furthermore, the increase in the average pre - edge energy upon increasing the coordination number is most likely attributed to destabilization of the d manifold upon increasing the number of ligands. similar effects on the pre - edge energies of the first row tm have previously been reported. in order to obtain a deeper understanding of the observed modulations in ca k - pre - edge intensities and energies, a td - dft approach was developed and calibrated to the experimental spectra. owing to the limitations of dft to accurately estimate the energies of the transition probabilities dominating the ca k - pre - edge spectra, an empirical, element specific shift should be determined. in fact, the calculated absolute transition energies carry large but highly systematic errors that arise from shortcomings of the density functionals in the core region, limitations of the one - particle basis set, and shortcomings in the accurate modeling of spin - free relativistic effects. given their highly systematic nature, all of these factors can (for a given basis set and density functional) be taken into account by introducing an element - dependent shift. in fact, it has been shown that a simple linear regression is sufficient to establish predictive accuracy in the calculated transition energies for any given element. this calibration needs to be carried out with respect to a test set of well known systems and has already been reported for metal and ligand k - edges in the framework of scalar relativistic dft methodology. the calibration of the ca k - pre - edge spectra was performed for a series of 12 structurally characterized compounds. in all cases, good agreement between the calculated versus experimental pre - edge intensities and energies was observed (figure 3, table 1). the intensity correlation resulted in a linear relationship with an r value of 0.968, while the energy correlation is also linear with an r value of 0.876. deviations may be attributed to errors intrinsic to data processing (figure 3) and limitations of the theoretical models. for instance, while they are likely to be small, possible vibronic contributions are not accounted for. similarly, complexes of intermediate symmetry such as the eight coordinate d2d / d4d complex 8 may have a geometry optimized structure which does not fully capture the interplay between the limiting symmetries again causing deviations in calculated values from experiment. however, both the intensity and energy correlation values for goodness of fit are consistent with those of previously reported td - dft calculated xas spectra and accurately predict the experimental trends observed as illustrated below. from the calibration fits, the energy shift to be applied to the calculated transition energies was found to be 50.17 0.08 ev, with an intensity normalization factor of 3500 300. figure 4 shows an overlay of representative experimental ca k - pre - edges, their corresponding fits, and the td - dft calculated transitions. difference density maps were used to visualize the transitions in terms of a molecular orbital picture (figure 5). the maps consist of a difference in electron density between the excited state and ground state and shows the shift in electron density due to the excitation. as the ca 1s orbital is highly localized and compact, the difference density map will be dominated by the contribution from the acceptor orbital. from this analysis, it is evident that the ca k - pre - edge consists of 1s d excitations (figures 4 and 5). in contrast to the first transition series, one generally does not consider a dominant 3d contribution to bonding in ca. nevertheless, the 3d orbitals are unoccupied and thus have xas transitions, which may include both quadrupole (1s 3d) and dipole allowed (1s 3d + np) transitions. it is the dipole allowed contribution that is responsible for the increase in pre - edge intensities over the present series. pre - edges of ca k - edge with their associated td - dft transitions and acceptor orbital assignment. from top to bottom : 7, 8, 7, 6. spectral fitting : rising edge (dotted lines), pre - edges (dashed lines). (right) breakdown of contributions to the total calculated transition intensities in terms of total dipole (green) and quadrupole (red) transition intensities, summed over the whole pre - edge. the calculated transition energies were shifted by 50.17 ev, and intensities were multiplied by a factor of 3500. difference density maps for 1s 3d transitions in 6. at the six coordinate pseudo - octahedral limit (figure 4, 6), the pre - edge consists of two small features due to quadrupole transitions. the calculated and experimental spectra both show a 1 ev split with transitions roughly grouped into the t2 g and eg set of acceptor orbitals. this is due to destabilization of the dz and dx y orbitals upon interaction with the ligands (figure 5). for the seven and eight coordinate complexes, there is an increase in intensity concomitant with the presence of dipole character. in the seven coordinate noncubane complex (7), with an approximate c2v local symmetry at ca, p d mixing is formally allowed for all of the d orbitals except dxy, which has the lowest calculated intensity. on the other hand, in the pseudo d2d eight coordinate complex 8, p d mixing is lowest in the dz and dx y orbitals, consistent with expectations from group theory. seven coordinate 7 species, where the calcium center has a c3v local symmetry that favors p d mixing in all the d orbitals. an in silico study of a hexa - aqua calcium complex, [ca(h2o)6 ], was carried out in order to further understand the nature of p d mixing responsible for intensity variations at the ca k - pre - edge. a c4v distortion was applied to a model octahedral [ca(h2o)6 ] complex, with initial ca oh2 distances of 2.4, by varying the distance of one of the ca both compression and elongation of the ca oh2 axial bond are expected to enhance p d mixing, through symmetry lowering and thus increase the pre - edge intensity. when the calculated intensities for all transitions in the pre - edge region are summed (black line, figure 6), one observes that the maximum pre - edge intensity occurs upon the greatest compression or greatest elongation of the ca oh2 bond rather than an elongation, which may be in part explained by covalency arguments, as has been previously discussed. however, if one sums the ca p - character contributing to all acceptor molecular orbitals over the pre - edge region (green line, figure 6), one finds that the percent p - character does not directly correlate with the intensity increase, as one might naively expect. this can be rationalized in terms of the origins of the p - character. depending on the length of the ca ligand bond, in principle, both 3p and 4p orbitals can contribute, with 3p orbitals having larger contributions at shorter distances. it is important to recognize, however, that the 3p and 4p orbitals will have different intrinsic dipole moment integrals. a 1s 3p dipole transition is expected to be significantly more intense than a 1s 4p transition, as the 3p orbitals have far more contracted radial functions, resulting in a larger intrinsic transition dipole moment integral. (top) variation in pre - edge intensity (black) and p - character (green) ; (bottom) variation in 3p (gray) and 4p (cyan) orbital energies. it is then reasonable to discuss the changes in intensity as coming from p d mixing with the p - character due to a combination of 3p and higher energy np orbitals. based on this hypothetical series, it is predicted that as the axial ca - aqua bond distance is compressed, the 3p and 4p orbitals are closer in energy, and when the bond is elongated, their energies become more separated, with the 3p orbitals being largely responsible for the energy difference (figure 6, bottom). as the 3p and higher np orbitals become energetically closer, more 3p mixing into these orbitals is possible. consequently as 3p mixing increases, less total p character is required for an increase in pre - edge intensity (figure 6, top). thus far, the current study has shown a correlation between ca k - pre - edges and ca coordination geometry by comparing structurally characterized complexes with their experimental spectra. it is important, however, to be able to deduce structure function relationships from experimental spectra even in the absence of a priori structural information. compounds 7 and 7 together with its one electron reduced analog 7 are good structural references for the mn3o4ca cubane - like part of the core of the oec cluster. these models offer an ideal test set for exploring the correlations between spectroscopic characteristics and geometric and electronic structures with relevance to the heterometallic cluster of the oec. cyclic voltammetry studies show that the one electron mn centered reduction of 7 (mn3o4ca) to 7 (mnmn2o4ca) occurs as a quasireversible wave at 890 mv versus ferrocene / ferrocenium in dmf. however, while the structure 7 was characterized via crystallography, the structure for 7 was not. therefore, a model was inferred for 7 based on the previously published analog, containing a sc center instead of ca. calculating the xas spectrum, and comparing it to the experimentally determined 7 spectrum, supports this model s validity and demonstrates the ability of ca k - edge xas to provide structural information. experimentally, the 7 cubane - like complex has a distinct pre - edge signature at the ca k - edge. a one electron chemical reduction leading to the 7 species results in a ca k - edge spectrum with a 16 3% lower pre - edge intensity (table 1, figure 7). a similar change in intensity is not observed in the noncubane complexes 7 and 7, which have similar pre - edge intensities. this implies that factors other than electronegativity are the driving force for the changes in intensity. complex 7 has a favorable geometry in terms of p d mixing ; however this is expected to change upon one electron reduction. indeed, a jahn teller like distortion (jt) is predicted to occur at the mn(iii) center in the 7 model, resulting in subtle changes in both ca bond distances and bond angles. the result is a calculated ca k - pre - edge with a 23% lower intensity in the reduced species 7 than in its oxidized counterpart (7 ; figure 7). furthermore, the effect of total spin at the mn centers on the calculated ca k - pre - edge was explored. the xas spectra for a high - spin (s = 5) system, as well as mn(iii)mn(iv) (s = 1) and mn(iv)mn(iv) (s = 2) antiferromagnetically coupled systems were calculated, and the spin state was found not to have any significant impact on the ca k - pre - edge (figure 7). similarly, previous work by pantazis. used an in silico truncated model of 7 to investigate the possible reduction of each of the mn(iv) centers to mn(iii) and the resulting preferred jt distortion. three lowest energy structural models for the reduced 7 were proposed, of which two were high - spin (s = 5) and one was of intermediate spin (s = 2). calculation of ca k - edge spectra for these models all resulted in a 25% decrease in intensity for the pre - edge, independent of spin state, comparable to the result from using the 7 model presented herein (supporting information figure s5). experimental spectra (top). calculated spectra for 7 and 7 (bottom). a 50.17 ev energy shift was applied, and intensities were multiplied by 3500 units. a series of six, seven, and eight coordinate calcium compounds were systematically investigated using ca k - edge xas. the spectra of six coordinate complexes are distinguished by a low intensity pre - edge split by 1 ev corresponding to excitations from the 1s orbital to the t2 g and eg set of 3d orbitals. seven and eight coordinate species consist of a single well - resolved 1s 3d feature in the pre - edge with seven coordinate mn3o4ca cubane - like (7, 7) clusters having the most intense pre - edges. a td - dft based protocol was developed to calculate and analyze the ca k - pre - edges. it was found that the determining factor for the pre - edge is the calcium coordination number and geometry. the intensities of the transitions are dependent on p d mixing, which is least favored in six coordinate centro - symmetric species and becomes more favored as the symmetry is lowered in seven and eight coordinate complexes. at the c3v limit, which best characterizes the cubane - like complexes, the highest pre - edge intensities are observed. the distribution of the transition energies correlates with an energetically destabilizing interaction with the ligands, where 3d orbitals interacting with the ligands are higher in energy. furthermore, we have investigated the potential applicability of ca k - edge xas to probe changes in the mn4o5ca cluster of the oec as it progresses through the kok cycle. such investigations could provide important complementary information to existing mn xas data, while offering a more streamlined analysis due to the presence of only one ca in the cluster, as opposed to four mn ions. it is already evident that ca xas is sensitive to the ligand coordination environment directly surrounding the ca center in terms of coordination number and ligand geometry. since calcium is proposed to play a direct role in the catalytic cycle by binding one substrate water molecule, ca k - edge xas could potentially make an important contribution in further understanding the mechanism of water oxidation. however, the question arises if xas at the ca center of the oec is sensitive to global changes of the cluster. using the oec closed cubane cluster mimics, 7 and its one electron reduced analog 7, the sensitivity of ca xas to a one electron reduction at one of the three mn centers was investigated. it was found that reduction of one mn(iv) to mn(iii) produces a jt distortion at the mn, which can be detected through changes in the ca k - pre - edge xas data. these studies thus establish ca xas as a potentially powerful probe for the understanding of the geometric and electronic structural changes that occur at the oec. for example, ca k - pre - edge xas could be used to differentiate between the proposed oec s2 state conformations that consist of the open - cubane form with an epr signal centered at g 2.0 and a closed - cubane form with an epr signal centered at g 4.1. the closed - cubane s2 state model has a cubane - like mn3o4ca structural motif, analogous to the cubane - like model series presented herein, connected to the remaining dangling mn by a mn o mn oxo bridge. conformation on the other hand has a structurally distorted cubane motif where the calcium forms an oxo bridge with the dangling mn. the current xas study strongly suggests that there should be distinct differences in the ca k - pre - edges of the two models, and therefore the application of these methods to the s states of the oec are the subject of ongoing research in our laboratories.	herein, ca k - edge x - ray absorption spectroscopy (xas) is developed as a means to characterize the local environment of calcium centers. the spectra for six, seven, and eight coordinate inorganic and molecular calcium complexes were analyzed and determined to be primarily influenced by the coordination environment and site symmetry at the calcium center. the experimental results are closely correlated to time - dependent density functional theory (td - dft) calculations of the xas spectra. the applicability of this methodology to complex systems was investigated using structural mimics of the oxygen - evolving complex (oec) of psii. it was found that ca k - edge xas is a sensitive probe for structural changes occurring in the cubane heterometallic cluster due to mn oxidation. future applications to the oec are discussed.
oral cancer ranks from 6 to 8 as the most common cancer in the world. cancer presently is the second most common cause of morbidity and mortality in the world after cardiovascular problems. cancers are heterogeneous cellular entities whose growth is dependent on reciprocal interactions between genetically altered cells and the microenvironment in which they reside. the microenvironment or more commonly referred as the tumor stroma is required for nutritional support and for the removal of waste products. it comprises of connective tissue, blood vessels, innate and adaptive immune cells, etc. innate immune cells include granulocytes, dendritic cells, macrophages, natural killer (nk) cells and mast cells. nk cells play a critical role both in innate immunity and adaptive immunity through cytokine secretion or by direct interaction with dendritic cells. nk cells provide resistance to pathogens and facilitate tumor immunosurveillance by two mechanisms which involve cytokine release interferon gamma (ifn-) and perforin - dependent target cell elimination. thus, this study was an attempt to analyze the presence of nk cells which is an integral part of tumor stroma and its effect on tumor progression. one hundred histologically diagnosed samples of oral squamous cell carcinoma (oscc) of various grades were retrieved for a period of year 20072010 from the archives of department of oral pathology and microbiology. all the samples were subdivided into two groups, those who were dead : group i and those who were alive : group ii. each patient 's relative was called every 6 month and was asked about the status of the patient. at the last follow - up, taken after approximately 3 years after surgery, 56 of 100 (56%) patients were alive and disease free, 5 (5%) patients were alive with recurrence of disease and 39 (39%) patients died of the disease. the paraffin - embedded tissue section of 4 m was obtained from archival tissues which were stained for routine hematoxylin and eosin and for the expression of cd57 by immunohistochemistry (ihc). sections were hydrated with increasing grades of alcohol and brought to distilled water and treated with hydrogen peroxide (h2o2) to eliminate endogenous peroxidase activity. then, antigen retrieval with tri - sodium citrate for cd 57 (clone tb01, lot # 00081714, primary antibody monoclonal mouse antihuman antibody the tissue was incubated sequentially with primary antibody cd57, which binds to specific tissue antigen on mononuclear inflammatory cells. dako envision system hrp, labeled polymer detection system and 3,3'-diaminobenzidine substrate solution (dab) was used that resulted in the formation of a colored precipitate at the tissue antigen binding sites. visualization was aided by counter staining with hematoxylin. in each slide, 5 high power fields (hpf) were selected. in each hpf, cytoplasm of the cells stained with cd57, regardless of the intensity of the stain were counted as positive. the total number of positive cells was obtained by addition of total number of positive cells in each hpf. to obtain labeling index, the sections stained with cd57 were examined under leica dmlb2 (leica microscope) at 40x. the positive control was examined for the presence of the colored end product at the site of the target antigen (dab chromogen brown end product). the absence of the colored end products and nonspecific staining in negative control confirmed the specificity of the primary antibody. cells were considered positive for cd57 if there was intracytoplasmic dab staining (chromogen color). all lymphoproliferative cells whose cytoplasm stained brown were scored positive regardless of intensity of staining. one hundred histologically diagnosed samples of oral squamous cell carcinoma (oscc) of various grades were retrieved for a period of year 20072010 from the archives of department of oral pathology and microbiology. all the samples were subdivided into two groups, those who were dead : group i and those who were alive : group ii. each patient 's relative was called every 6 month and was asked about the status of the patient. at the last follow - up, taken after approximately 3 years after surgery, 56 of 100 (56%) patients were alive and disease free, 5 (5%) patients were alive with recurrence of disease and 39 (39%) patients died of the disease. the paraffin - embedded tissue section of 4 m was obtained from archival tissues which were stained for routine hematoxylin and eosin and for the expression of cd57 by immunohistochemistry (ihc). sections were hydrated with increasing grades of alcohol and brought to distilled water and treated with hydrogen peroxide (h2o2) to eliminate endogenous peroxidase activity. then, antigen retrieval with tri - sodium citrate for cd 57 (clone tb01, lot # 00081714, primary antibody monoclonal mouse antihuman antibody the tissue was incubated sequentially with primary antibody cd57, which binds to specific tissue antigen on mononuclear inflammatory cells. dako envision system hrp, labeled polymer detection system and 3,3'-diaminobenzidine substrate solution (dab) was used that resulted in the formation of a colored precipitate at the tissue antigen binding sites. in each slide, 5 high power fields (hpf) were selected. in each hpf, cytoplasm of the cells stained with cd57, regardless of the intensity of the stain were counted as positive. the total number of positive cells was obtained by addition of total number of positive cells in each hpf. to obtain labeling index, the sections stained with cd57 were examined under leica dmlb2 (leica microscope) at 40x. the positive control was examined for the presence of the colored end product at the site of the target antigen (dab chromogen brown end product). the absence of the colored end products and nonspecific staining in negative control confirmed the specificity of the primary antibody. cells were considered positive for cd57 if there was intracytoplasmic dab staining (chromogen color). all lymphoproliferative cells whose cytoplasm stained brown were scored positive regardless of intensity of staining. cd57 li : cd57 was expressed in oscc in decreasing order from well differentiated to poorly differentiated scc [figures 13 ]. on correlating cd57 labeling index with status of life (either dead or alive) of patients with oscc using spearman 's correlation coefficient, p value was found to be significant (p = 0.00). when pearson 's linear correlation was used, then also p value was significant (p 0.0001). well - differentiated squamous cell carcinoma showing cd57 + cells within mononuclear inflammatory cell infiltrate in the tumor stroma (ihc stain, 400) moderately differentiated squamous cell carcinoma showing cd57 + cells within mononuclear inflammatory cell infiltrate in the tumor stroma (ihc stain, 400) poorly differentiated squamous cell carcinoma showing cd57 + cells within mononuclear inflammatory cell infiltrate in the tumor stroma (ihc stain, 400) carcinomas are malignant neoplasms derived from epithelial cells and represent the most common form of human cancer. oral cancer ranks from 6 to 8 as the most common cancer in the world. these studies have focused mostly on the functions of oncogenes and tumor suppressor genes, and the signaling pathways regulating cell proliferation and/or cell death. however, such studies have informed us only about one aspect of tumor environment ignoring the fact that cancers are heterogeneous cellular entities whose growth is dependent on reciprocal interactions between genetically altered cells and the microenvironment in which they reside. the microenvironment or more commonly referred as the tumor stroma is required for nutritional support and for the removal of waste products. the tumor stroma comprises connective tissue, blood vessels, innate and adaptive immune cells, etc. innate immune cells include granulocytes, dendritic cells, macrophages, nk cells and mast cells. nk cells play a critical role both in innate immunity and adaptive immunity through cytokine secretion or by direct interaction with dendritic cells. immune system manipulation against the tumor cell is multifactorial. the immune defense against tumor cell is mediated initially by the innate immune cells, i.e., nk cells, nk t cells, cytokines and complement proteins, and later by adaptive immune system (b and t cells). nk cells are one of the most important effector t - lymphocytes with an effective antitumor effect.. cell surface expression of major histocompatibility complex (mhc) class i is often downregulated by tumors and virally infected cells. the 3-year survival in patients with oscc who were treated with surgical resection was about 56% patients, 5% patient survived but with recurrence and 39% patients were dead. they documented that 3555% of patients with head and neck squamous cell carcinoma remained disease free after 3 years post standard treatment. mortality in the patient with oscc followed up over a period of 3 years could be due to recurrence or delay in treatment. the presence of nk cells in oscc patients was evaluated in this study with ihc. this is the first study in which a labeling index of nk cells in oscc was calculated. earlier studies were semi - quantitative and qualitative in which expression of cd57 was evaluated either in terms of positivity or negativity, intensity of staining or a definite number of cells / hpf. however, in the present study, cd57 labeling index was calculated, i.e., number of positive cells / hpf was evaluated. after extensive research of data, we found that this was the first documented instance where the cd57 labeling index was calculated. the labeling index was less in dead patients, whereas the labeling index in patients who had survived was more. the mean labeling index was 3.67 and 10.67 in dead and alive patients, respectively. due to lack of similar study in oscc, we could not carry out any direct comparisons, and hence, we have made comparison with the results documented for other carcinomas. ishigami. carried out a similar study on gastric carcinoma cases and showed that more number of nk cell infiltrate in patients showed better prognosis than those showing less nk cell infiltrate. villegas. also carried out similar study in lung squamous cell carcinoma concluding that patients with less nk cell infiltrate showed worse prognosis than those showing more nk cell infiltrate. the positive correlation of cd57 with survival in tumor stroma may be due to the fact that nk cells play a major role in eliminating neoplastic cells. during the course of tumor establishment it has been well documented that in oscc, there is either downregulation or lack of mhc class i protein expression. loss of mhc class i molecule is an effective way of evading host immunosurveillance other than nk cells. this activates nk cells, as nk cells does not require mhc class i expression by the target cells. nk cells possess nk receptors, i.e., activatory and inhibitory nk cell receptor (nkr). killer inhibitory receptor molecules recognize mhc class i and when they engage their ligand, nk cell - mediated lysis is inhibited. therefore, a cell devoid of mhc class i expression may be able to evade specific cytotoxic t - lymphocytes recognition but would remain a target for nk cells. nkg2d ligand binds to nkg2d ligand - specific protein, transmits activation signal and induces immune effector cells to clear tumor cells. around 6% of samples showed negative staining for cd57, among them 3% were dead and 3% were alive. the present study was aimed at the correlation of the expression of cd57 with 3 years survival in patients with oscc. this study revealed that there was a significant correlation of cd57 labeling index with the status of life. as the cd57 labeling index increased, it was most likely that the patient was alive. the mean cd57 labeling index was much more in live patients as compared to the dead one. that risk of overall short survival was 2.5 fold higher in patients with low tumor infiltration by cd57 + lymphocytes. there are similar studies conducted on prostate carcinoma, colorectal carcinoma and lung carcinoma by liu., coca. and villegas. respectively, who also documented that increase in tumor infiltration by cd57 + lymphocytes results in a better prognosis. evidence exists that growth and metastatic growth of tumor are dependent on their capacity to evade host immune surveillance and overcome host defense. all tumors express antigens, but in many instances, an inadequate immune response is seen. the antitumor response by the immune inflammatory cells in the tumor microenvironment is usually imparted by cytotoxic t - lymphocytes and nk cells. the association of increase survival with increase in cd57 expression could be due to : cytotoxic t - lymphocytic response is mhc class i restricted as their activation is dependent on mch class i expression on target cells. however, there are various studies in the literature which have stated reduction or absence of mhc class i molecule expression by neoplastic cells. tumors with downregulated classical mhc class i expression allows them to escape cytotoxic t - lymphocytes immunosurveillance. thus, nk cells play a major role as a cytotoxic cell for those tumor cells that have lost mhc class i expression.nk cells are also known to regulate hematopoiesis and antibody production by b cells. thereby increasing immunosurveillance.early appearing, tumor infiltrating nk cells play a crucial role in the generation of antitumor t lymphocytes. there is a possibility that nk cells have an influence on generation of antitumor cytotoxic t - lymphocytes through production of ifn-. this cytokine environment is important for the development of antigen - specific cd4 + and cd8 + t - cells. furthermore, it is well documented that ifn- upregulates the expression of mhc class i and mhc class ii molecule. thus, the upregulation of mhc class ii on macrophages and mhc class i on tumor cell allow t - cells to recognize tumor - specific antigen.nk cells do not have tcrs on the cellular membrane, and their activation does not require mhc class i expression by the target cells. nk cells express a surface receptor that is nkr that can be classified as inhibitory nkr and activatory nkr. inhibitory nkrs are kir and cd94-nkg2a / b and activatory nkrs are nkg2d, dnam-1 and natural cytotoxicity receptors. these inhibitory kir and cd94-nkg2a / b receptors are responsible for recognition of different alleles of mhc class i molecules. the lack of even a single mhc class i sensitize them to nk cell cytotoxicity. the activatory nkr triggers spontaneous and potent cytotoxicity.evidence shows a link between tumorigenesis, the dna damage response and the immune response. dna damaging agents or dna lesions associated with tumorigenesis activate the dna damage response in damage cells. this response results in upregulation of nkg2d ligand which stimulates the nk cells to attack the diseased cells.nk cells express low affinity for immunoglobulin g receptor cd16 (activatory nkr), which enables them to recognize and kill target cells opsonized with antibodies by antibody dependent cell - mediated cytotoxicity. cytotoxic t - lymphocytic response is mhc class i restricted as their activation is dependent on mch class i expression on target cells. however, there are various studies in the literature which have stated reduction or absence of mhc class i molecule expression by neoplastic cells. tumors with downregulated classical mhc class i expression allows them to escape cytotoxic t - lymphocytes immunosurveillance. thus, nk cells play a major role as a cytotoxic cell for those tumor cells that have lost mhc class i expression. nk cells are also known to regulate hematopoiesis and antibody production by b cells. thereby increasing immunosurveillance. early appearing, tumor infiltrating nk cells play a crucial role in the generation of antitumor t lymphocytes. there is a possibility that nk cells have an influence on generation of antitumor cytotoxic t - lymphocytes through production of ifn-. this cytokine environment is important for the development of antigen - specific cd4 + and cd8 + t - cells. furthermore, it is well documented that ifn- upregulates the expression of mhc class i and mhc class ii molecule. thus, the upregulation of mhc class ii on macrophages and mhc class i on tumor cell allow t - cells to recognize tumor - specific antigen. nk cells do not have tcrs on the cellular membrane, and their activation does not require mhc class i expression by the target cells. nk cells express a surface receptor that is nkr that can be classified as inhibitory nkr and activatory nkr. inhibitory nkrs are kir and cd94-nkg2a / b and activatory nkrs are nkg2d, dnam-1 and natural cytotoxicity receptors. these inhibitory kir and cd94-nkg2a / b receptors are responsible for recognition of different alleles of mhc class i molecules. single nk cell comprises numerous kir. the lack of even a single mhc class i sensitize them to nk cell cytotoxicity. evidence shows a link between tumorigenesis, the dna damage response and the immune response. dna damaging agents or dna lesions associated with tumorigenesis activate the dna damage response in damage cells. this response results in upregulation of nkg2d ligand which stimulates the nk cells to attack the diseased cells. nk cells express low affinity for immunoglobulin g receptor cd16 (activatory nkr), which enables them to recognize and kill target cells opsonized with antibodies by antibody dependent cell - mediated cytotoxicity. despite all the ways in which the nk cell recognize target cell, secretory lysozyme exocytosis and perforin - dependent target cell elimination are required. this is divided into four stages : activating, lytic immunologic synapse forms at the point of contact with the target cell, resulting in rearrangement of the actin cytoskeletonmicrotubules organizing center of the nk cells and secretory lysosomes are polarized toward the lytic synapsesecretory lysosomes dock with the plasma membrane at the lytic synapse, followed byrelease of cytotoxic content. activating, lytic immunologic synapse forms at the point of contact with the target cell, resulting in rearrangement of the actin cytoskeleton microtubules organizing center of the nk cells and secretory lysosomes are polarized toward the lytic synapse secretory lysosomes dock with the plasma membrane at the lytic synapse, followed by release of cytotoxic content. thus, we conclude that increase in the expression of cd57 in the tumor stroma of oscc may serve as a good prognostic marker for the patients. department of oral and maxillofacial pathology and microbiology, sharad pawar dental college, wardha. department of oral and maxillofacial pathology and microbiology, sharad pawar dental college, wardha.	objectives : natural killer (nk) cells are important effector lymphocytes. nk cells are considered to represent innate immune system. nk cells target and kill aberrant cells such as virally infected and tumorigenic cells. the purpose of this study was to assess the expression of cd57 in oral squamous cell carcinoma (oscc) and to correlate the expression of cd57 with 3 years survival in patients with oscc.materials and methods : about 100 histopathologically diagnosed cases of oscc of various grades were divided into two groups, i.e., group i (dead patients) and group ii (live patients) from the archives of department of oral pathology and microbiology. cd57 was detected in these tissues by immunohistochemistry.result:the results were analyzed using spearman 's correlation coefficient and students unpaired t - test. the mean cd57 labeling index in group ii was significantly higher than that found in group i (p = 0.000). there was a significant correlation (p = 0.00) in the mean cd57 levels between groups i and ii and prognosis of patient.conclusion:cd57 could be a good prognostic marker for oscc patients.
male c57bl6jolahsd mice were purchased from harlan netherlands (harlan, horst, the netherlands), and male il-6 ko mice and respective wild - type mice were purchased from charles river laboratories (wilmington, ma). all mice were housed in a pathogen - free environment on a 12-h light - dark cycle, with free access to standard rodent diet (provimi kliba, kaiseraugst, switzerland). all protocols conformed to the swiss animal protection laws and were approved by the cantonal veterinary office in zurich, switzerland. mice were anesthetized with isoflurane (abbott, baar, switzerland). both epididymal fat pads (average size per pad 75 mg [range 6590 mg ]) were removed from the donor mouse, rinsed with 0.9% saline, and thereafter stitched to the visceral side of the peritoneum (caval transplantation) or to the mesenterium of the recipient mouse (portal transplantation) using monocryl 6.0 (johnson - johnson, spreitenbach, switzerland). sham - operated control mice underwent the same surgical procedure without receiving a fat transplant. subcutaneous injection of buprenorphine every 12 h for 2 days was used for analgesia. to prevent transplant rejection, glucose (2 g / kg body wt) was injected intraperitoneally and blood was collected from the tail vein at different time points, as indicated (5). plasma glucose was measured using a glucose meter (ascensia contour ; bayer, zurich, switzerland). sections were obtained at the time of death and stained with hematoxylin and eosin. for each fat pad of a mouse, at least 100 adipocytes were analyzed. images were analyzed using imagej software for quantification (national institutes of health, bethesda, md). mac2-positive cells were counted per nuclei of seven randomly selected fields per section containing at least 90 cells. clamps were performed in freely moving mice 5 weeks after transplantation as described previously (7). glucose infusion rate was calculated once glucose infusion reached a more or less constant rate with blood glucose levels at 5 mmol / l. thereafter, blood glucose was kept constant at 5 mmol / l for 20 min (with no or minimal adjustment of the glucose infusion rate), and glucose infusion rate was calculated. the glucose disposal rate was calculated by dividing the rate of [3-h]glucose infusion by the plasma [3-h]glucose specific activity (8,9). endogenous glucose production during the clamp was calculated by subtracting the glucose infusion rate from the glucose disposal rate (8,9). insulin - stimulated glucose disposal rate was calculated by subtracting basal endogenous glucose production (equal to basal glucose disposal rate) from glucose disposal rate during the clamp (10). plasma adiponectin, leptin, and il-6 levels were determined with mouse lincoplex kits from linco research (labodia, yens, switzerland) ; plasma insulin and ffa levels were measured as previously described (5). liver tissue (30 mg) was homogenized in pbs, and lipids were extracted in a chloroform - methanol (2:1) mixture. total liver lipids were determined by a sulfophosphovanillin reaction as previously described (7). total rna from fat pads was extracted with the rneasy lipid tissue mini - kit (qiagen). a total of 0.75 g rna was reverse - transcribed with superscript iii reverse transcriptase (invitrogen) using random hexamer primer (invitrogen). relative gene expression was obtained after normalization to 18srna (applied biosystems), using the formula 2 (11). the following primers were used : tnf- mm004483258_m1, il-6 mm00446190_m1, kc mm00433859_m1, il-1 mm0043422/_m1, mcp-1 mm00441242_m1, fas mm00433237_m1, fasl mm00438864_m1, cd11c mm00498698_m1, hif-1 mm00468869_m1, socs.3 mm 00545913_s1, cd68 mm03047343_m1, f4/80 mm00802529_m1, and cd11b mm00434455_m1 (applied biosystems). the following antibodies were used : anti phospho - akt (s473) and anti - akt were purchased from cell signaling ; secondary horseradish peroxidase conjugated antibody were purchased from either santa cruz biotechnology or alexis biochemicals. data are presented as means sem and were analyzed by student t test or anova with a turkey correction for multiple group comparisons. male c57bl6jolahsd mice were purchased from harlan netherlands (harlan, horst, the netherlands), and male il-6 ko mice and respective wild - type mice were purchased from charles river laboratories (wilmington, ma). all mice were housed in a pathogen - free environment on a 12-h light - dark cycle, with free access to standard rodent diet (provimi kliba, kaiseraugst, switzerland). all protocols conformed to the swiss animal protection laws and were approved by the cantonal veterinary office in zurich, switzerland. mice were anesthetized with isoflurane (abbott, baar, switzerland). both epididymal fat pads (average size per pad 75 mg [range 6590 mg ]) were removed from the donor mouse, rinsed with 0.9% saline, and thereafter stitched to the visceral side of the peritoneum (caval transplantation) or to the mesenterium of the recipient mouse (portal transplantation) using monocryl 6.0 (johnson - johnson, spreitenbach, switzerland). sham - operated control mice underwent the same surgical procedure without receiving a fat transplant. subcutaneous injection of buprenorphine every 12 h for 2 days was used for analgesia. to prevent transplant rejection, for the intraperitoneal glucose tolerance test, mice were fasted overnight. glucose (2 g / kg body wt) was injected intraperitoneally and blood was collected from the tail vein at different time points, as indicated (5). plasma glucose was measured using a glucose meter (ascensia contour ; bayer, zurich, switzerland). sections were obtained at the time of death and stained with hematoxylin and eosin. for each fat pad of a mouse, at least 100 adipocytes were analyzed. images were analyzed using imagej software for quantification (national institutes of health, bethesda, md). mac2-positive cells were counted per nuclei of seven randomly selected fields per section containing at least 90 cells. clamps were performed in freely moving mice 5 weeks after transplantation as described previously (7). glucose infusion rate was calculated once glucose infusion reached a more or less constant rate with blood glucose levels at 5 mmol / l. thereafter, blood glucose was kept constant at 5 mmol / l for 20 min (with no or minimal adjustment of the glucose infusion rate), and glucose infusion rate was calculated. the glucose disposal rate was calculated by dividing the rate of [3-h]glucose infusion by the plasma [3-h]glucose specific activity (8,9). endogenous glucose production during the clamp was calculated by subtracting the glucose infusion rate from the glucose disposal rate (8,9). insulin - stimulated glucose disposal rate was calculated by subtracting basal endogenous glucose production (equal to basal glucose disposal rate) from glucose disposal rate during the clamp (10). plasma adiponectin, leptin, and il-6 levels were determined with mouse lincoplex kits from linco research (labodia, yens, switzerland) ; plasma insulin and ffa levels were measured as previously described (5). liver tissue (30 mg) was homogenized in pbs, and lipids were extracted in a chloroform - methanol (2:1) mixture. total liver lipids were determined by a sulfophosphovanillin reaction as previously described (7). total rna from fat pads was extracted with the rneasy lipid tissue mini - kit (qiagen). a total of 0.75 g rna was reverse - transcribed with superscript iii reverse transcriptase (invitrogen) using random hexamer primer (invitrogen). relative gene expression was obtained after normalization to 18srna (applied biosystems), using the formula 2 (11). the following primers were used : tnf- mm004483258_m1, il-6 mm00446190_m1, kc mm00433859_m1, il-1 mm0043422/_m1, mcp-1 mm00441242_m1, fas mm00433237_m1, fasl mm00438864_m1, cd11c mm00498698_m1, hif-1 mm00468869_m1, socs.3 mm 00545913_s1, cd68 mm03047343_m1, f4/80 mm00802529_m1, and cd11b mm00434455_m1 (applied biosystems). the following antibodies were used : anti phospho - akt (s473) and anti - akt were purchased from cell signaling ; secondary horseradish peroxidase conjugated antibody were purchased from either santa cruz biotechnology or alexis biochemicals. data are presented as means sem and were analyzed by student t test or anova with a turkey correction for multiple group comparisons. to estimate potential metabolic effects of an artificial increase in fat tissue mass drained uniquely into the portal system, epididymal fat pads of a regular diet fed donor mouse were transplanted to the mesenterium of a regular diet fed littermate mouse using a novel surgical approach. at the time of death (5 weeks after transplantation), transplanted fat pads were adherent to the mesenterium and well vascularized (fig. mice tolerated the surgical procedure and the fat transplantation well ; no adverse events were noted. to confirm venous drainage of transplanted fat pads to the portal vein, evans blue 1b, transplanted fat pads were stained with evans blue, as was endogenous mesenteric fat tissue, but not the caval - drained epididymal fat tissue, confirming the portal drainage of the mesenteric - transplanted fat tissue. both epididymal fat pads were removed from a donor mouse and stitched to the mesenterium (ptx) of a littermate recipient. a : six weeks after transplantation, the abdominal cavity was opened and the transplanted fat pad was visualized. b : to confirm portal drainage of transplanted fat pads, evans blue dye (5 mg / ml) was injected into the portal vein. c : intraperitoneal glucose tolerance test (2 g glucose / kg body weight) was performed 4 weeks after surgical procedure in mice receiving portal drained transplants (ptx ;), in mice receiving caval drained transplants (ctx ;), or in sham - operated mice () after an overnight fast. results are expressed as mean blood glucose concentration (left panel) or area under curve (auc) (right panel) sem of 5 (ptx), 16 (ctx), or 23 (sham) animals. p 70% higher in portal than systemic blood samples of mice receiving a ptx (fig. 5b). even though this difference was not significant, these data suggest increased delivery of il-6 to the liver of mice receiving a portal transplant, potentially originating from the transplanted fat pad. consistent with an increased delivery of il-6 to the liver, hepatic socs3 mrna expression, which is induced by il-6 (14,15), was enhanced in mice receiving a portal - transplanted fat pad (fig. 5c). to substantiate the potential contribution of il-6 to the induction of insulin resistance in portal - transplanted mice, epididymal fat pads of an il-6 knockout donor were transplanted into a wild - type recipient (ptx - il6ko). 6a, glucose tolerance of such transplanted mice was not impaired compared with sham - operated wild - type mice. moreover, the insulin - induced increase in akt phosphorylation in liver samples was completely maintained in these mice (fig. 6b) in contrast to mice receiving a ptx from wild - type mice (fig. 3c). in addition, hepatic socs-3 mrna expression was not increased, likely because of il-6 deficiency (fig. these results point toward a causative role for il-6 in the development of insulin resistance in portal - transplanted mice. both epididymal fat pads were removed from an il-6 knockout mouse (donor) and stitched to the mesenterium of a wild - type mouse (recipient ; ptx - il6ko). a : intraperitoneal glucose tolerance test (2 g glucose / kg body wt) was performed 4 weeks after surgical procedure in mice receiving portal transplants (ptx - il6ko ;) or in sham - operated mice (). b : insulin (1 u / kg) was injected intraperitoneally in overnight fasted mice 5 weeks after transplantation. liver samples were harvested 15 min later, and phosphorylation of akt at s473 was determined by western blotting. c : five weeks after transplantation, liver tissue was collected and real - time pcr was performed. the portal hypothesis proposes that the liver is directly exposed to ffas and cytokines increasingly released from visceral fat tissue into the portal vein of obese subjects, thus rendering visceral fat accumulation particularly hazardous for the development of hepatic insulin resistance and type 2 diabetes (2,3). in the present study, we used a fat transplantation paradigm to (artificially) increase fat mass depot specifically. in accordance with the portal hypothesis, we demonstrate that fat transplant localization through its venous drainage determines its effect on glucose homeostasis. when using a fat pad transplantation paradigm, several factors need to be taken into account. first, the source of the transplanted tissue, i.e., which fat depot is used, may impact the metabolic outcome. second, the functional outcome of the transplant may depend on a myriad of host factors, which include (but are not limited to) the location (and thus venous drainage) of the transplant. previous studies by different groups including our own have examined the effect of adipose tissue transplantation into the abdominal cavity (5,6,16). yet, whereas our group only used epididymal fat pads for transplantation, the other groups implemented both subcutaneous and epididymal fat pads (6,16). (6) observed no changes in glucose tolerance when epididymal fat pads were transplanted into the abdominal cavity. however, as discussed by the authors, fat pads were drained both to the caval as well as to the portal vein. similarly, we show here that such dual drainage of transplanted fat tissue may in fact result in no in glucose tolerance (fig. one potential explanation might be the fact that the beneficial metabolic effect of the systemically drained fat transplant (5) was balanced by the detrimental effect of the portal - drained transplant. on the other hand, the lack of deterioration in glucose homeostasis of double - transplanted mice or in the study by tran. we further used the ptx paradigm to determine potential mediators of hepatic insulin resistance in conditions with increased visceral adiposity. adipose tissue inflammation was previously correlated with hepatic insulin resistance, and il-6 was suggested as a potential candidate (7,13,17). indeed, il-6 treatment of hepatocytes induced insulin resistance in vitro (18), and it seems to be generally accepted that il-6 causes hepatic insulin resistance (19). particularly, il-6 was shown to activate the negative insulin - signaling regulator socs3 in c57bl/6j mice (14,15), resulting in reduced activation of phosphatidylinositol 3-kinase and akt (18). furthermore, il-6 plasma concentration was 50% greater in the portal vein than in systemic circulation in obese subjects, and portal vein il-6 concentration correlated directly with systemic c - reactive protein concentrations (20). accordingly, il-6 expression in portal - transplanted adipose tissue and il-6 levels in the portal vein of such transplanted mice were increased (fig. 5a and b), whereas portal il-6 concentration was not increased in caval - transplanted mice (supplementary figure s2). 5c) and, consistently, insulin - stimulated akt - phosphorylation was reduced in liver samples of mice receiving portal transplants (fig. both changes could be prevented in mice receiving transplants from il-6 knockout mice (fig. interestingly, il-6 mrna expression was also significantly increased in caval - transplanted fat pads compared with endogenous (epididymal) fat pads (fig. 5a), consistent with the transplanted fat depot being the source of the elevated portal il-6 levels. remarkably, fat pads transplanted to both sites were characterized by increased expression of proinflammatory cytokines (fig. potential explanations might be a transplant rejection reaction (even though recipient and donor mice were littermates of inbred colonies) or an inflammatory reaction due to transient transplant hypoxia. intriguingly, increased production of proinflammatory cytokines by the transplanted fat pads only deteriorated glucose metabolism when they were drained to the portal vein. moreover, mesenteric transplantation of peritoneal tissue that did not contain adipose tissue had no effect on glucose tolerance (supplementary figure s3). these data suggest that the transplantation procedure on its own does not deteriorate glucose homeostasis, for example, by altering the delivery of inflammatory mediators from the gastrointestinal tract. such a finding emphasizes the importance of fat tissue localization and thus vascular drainage and suggests that the increased production of proinflammatory cytokines as it is found in obesity is particularly detrimental when released into the portal circulation supporting the portal hypothesis (3). however, it needs to be emphasized that we examined an artificial system herein, and, even though increased expression of proinflammatory cytokines in adipose tissue of obese subjects was previously demonstrated (21), the findings presented here may not be extrapolated to the situation of mesenteric fat accumulation. therefore, we presently can not exclude that the impaired glucose tolerance observed in the portally transplanted mice may not be due to the additional adipose tissue per se but rather due to the increased inflammatory products related to the transplantation procedure. moreover, besides il-6, other factors may have contributed to the deterioration of glucose tolerance and hepatic insulin resistance. interestingly, lack of il-6 also reduced expression of other proinflammatory factors such as il-1 in adipose tissue (supplementary figure s4). in addition, reduced cd11c expression levels point toward reduced macrophage infiltration. rotter. (22) suggested that il-6 induce insulin resistance in concert with other cytokines that are also upregulated in adipocytes. especially il-6 and il-1 were shown to mediate the change of hepatic glucose metabolism, influencing the glucoregulatory action of insulin through their signal pathways involving protein phosphorylation (21,23). we previously reported that fas activation by fasl treatment induced secretion of il-6 in 3t3-l1 adipocytes (7). conversely, adipocyte - specific fas - knockout mice showed decreased il-6 expression in adipose tissue and reduced circulating il-6 levels (7). thus, fasl - induced fas activation might be responsible for increased il-6 secretion of portal - transplanted fat pads, ultimately inducing hepatic insulin resistance. in support of the latter, fasl expression was not reduced in portal - transplanted il-6deficient fat pads, suggesting that fas activation may be indeed upstream of il-6 release (supplementary figure s4). strikingly, hepatic insulin resistance in our model was not associated with hepatic steatosis (fig. 4b), since it is often observed in high - fat diet induced hepatic insulin resistance (12). these changes might be due to a direct effect of high - fat diet on liver metabolism or due to an increased release of ffas from (mesenteric) adipose tissue. consistent with the latter notion, ffa levels were found to be increased in high fat fed mice, which was not the case in our study. in conclusion, our results demonstrate that the metabolic fate of intra - abdominal fat tissue transplantation is determined by the delivery of inflammatory cytokines to the liver specifically via the portal system providing clear direct support for the portal hypothesis.	objectivethe portal hypothesis proposes that the liver is directly exposed to free fatty acids and cytokines increasingly released from visceral fat tissue into the portal vein of obese subjects, thus rendering visceral fat accumulation particularly hazardous for the development of hepatic insulin resistance and type 2 diabetes. in the present study, we used a fat transplantation paradigm to (artificially) increase intra - abdominal fat mass to test the hypothesis that venous drainage of fat tissue determines its impact on glucose homeostasis.research design and methodsepididymal fat pads of c57bl6/j donor mice were transplanted into littermates, either to the parietal peritoneum (caval / systemic venous drainage) or, by using a novel approach, to the mesenterium, which confers portal venous drainage.resultsonly mice receiving the portal drained fat transplant developed impaired glucose tolerance and hepatic insulin resistance. mrna expression of proinflammatory cytokines was increased in both portally and systemically transplanted fat pads. however, portal vein (but not systemic) plasma levels of interleukin (il)-6 were elevated only in mice receiving a portal fat transplant. intriguingly, mice receiving portal drained transplants from il-6 knockout mice showed normal glucose tolerance.conclusionsthese results demonstrate that the metabolic fate of intra - abdominal fat tissue transplantation is determined by the delivery of inflammatory cytokines to the liver specifically via the portal system, providing direct evidence in support of the portal hypothesis.
in this article, we draw on national social survey data to identify and report on the social determinants of wellbeing in the uk and we consider some of the challenges the findings present for social policy. in the first section, we review some of the latest developments in subjective wellbeing (swb) research, and we consider how swb is now being measured by the office for national statistics (ons) in the uk. in our empirical section, we develop bivariate and multivariate logistic regression models, as well as testing for interaction effects, in order to assess the socio - economic and demographic characteristics that help to predict wellbeing in the national population. we do so using four global measures of swb. finally, in the last section, we consider some of the potential social policy implications raised by the findings. before describing the study methods and results, and discussing their implications, we review the uk 's new measuring national wellbeing (mnw) programme. it is now widely accepted that traditional economic measures are necessary, but not sufficient, to reflect national wellbeing (stiglitz., 2010). in recent years, measures of swb have been refined to help monitor social progress and policy (taylor, 2011). governments around the world including the british government are increasingly concerned about the quality of life and the environment in which we live, as well as the traditional measures of gdp and economic growth that help to define living standards in society (stratton, 2010). the measurement of swb has advanced rapidly over the last two decades (diener, 2009a). researchers usually draw a basic distinction between self - reported wellbeing, i.e. swb, and the more objective non - self - reported assessments and measures (diener, 2009b). in this article, we are only interested in subjective measures. broadly speaking, as dolan and metcalfe (2012) maintain, there are three main theoretical strands underpinning the measurement of swb which are relevant to this study : the evaluative approach to swb asks individuals to reflect on their life and make a cognitive assessment of how their life is going overall, or on certain aspects of their life. life satisfaction, as used here, is dependent on a global appraisal of life.the hedonic approach seeks to measure people 's feelings and emotions as diener (2009a) observes. general states of happiness and anxiety are used in this study, which form part of the more global cognitive appraisal of wellbeing.the eudemonic approach, sometimes referred to as the psychological functioning or flourishing approach, draws on self - determination theory and taps into our sense of purpose and meaning in life, with notions of the worthwhile life employed here. evaluative approach to swb asks individuals to reflect on their life and make a cognitive assessment of how their life is going overall, or on certain aspects of their life. life satisfaction, as used here, is dependent on a global appraisal of life. the hedonic approach seeks to measure people 's feelings and emotions as diener (2009a) observes. general states of happiness and anxiety are used in this study, which form part of the more global cognitive appraisal of wellbeing. eudemonic approach, sometimes referred to as the psychological functioning or flourishing approach, draws on self - determination theory and taps into our sense of purpose and meaning in life, with notions of the worthwhile life employed here. in the classical philosophical tradition, interpretations of eudaimonia and human flourishing were defined by aristotle as the highest human good and included such things as spiritual fulfilment and civic virtue (bok, 2010). eudemonic wellbeing but there are of course other more objective measures of human wellbeing, of equality and human rights, and capability, being developed and refined for the development of social policy (dean, 2010). interest in the idea of national accounts for monitoring population wellbeing is growing (diener., 2009a) and swb measures and findings are increasingly being used to inform and appraise social policy (dolan and metcalfe, 2012). in the uk, the mnw programme was launched by the ons in 2010, as a response to the growing domestic as well as international policy imperative (table 1). the programme is designed to provide new statistical measures of swb, urgently needed to help monitor social progress and shape the direction of social policy. following the recommendations of dolan and metcalfe (2012), and those of stiglitz. (2010), ons now attempts to capture the three different components of swb in household surveys (ons, 2011a, 2011b, 2011c, 2012). table 1.key developments in measuring wellbeing1994 united nations publishes first human development index.2000 first issue of the journal of happiness studies is published.2002 uk cabinet office strategy unit report, life satisfaction : the state of knowledge and implication for government.2007 european commission initiates the beyond gdp project.2008 president sarkozy establishes the commission on the measurement of economic performance and social progress.2009 oecd starts better life initiative and work programme on measuring wellbeing and progress.2010 the us government establishes a commission on key national indicators, allocating $ 70 million to the project.2010 the uk office for national statistics begins a programme to develop statistics to measure national wellbeing.2011 the us national research council, the national institute on aging and the uk economic and social research council jointly support an expert panel on subjective wellbeing and public policy.2011 un general assembly resolution on happiness 65/309.2012 un high - level meeting on happiness and wellbeing. release of the un world happiness report.source : parliamentary office of science and technology (post) (2012 : 2). key developments in measuring wellbeing source : parliamentary office of science and technology (post) (2012 : 2). the ons have discussed their emerging survey findings on swb in various publications (e.g., ons, 2011a, 2011b). although their investigations are informative, the ons has, however, deliberately stopped short of any sophisticated analysis of data, including the sort of multivariate modelling that is required to shed greater light on the complexity of swb in the social world (byrne, 2011). to - date, ons reports have largely been descriptive, showing basic cross - tabulations and average estimates of swb for different sections of the population (as shown in table 2, for example). their findings do not reveal with any degree of certainty which sections of the british population are particularly vulnerable to experiencing low levels of swb. yet we know from the international research literature that a range of socio - demographics (e.g., age, gender, income, household composition, unemployment and disability) can help to explain wellbeing (e.g., clark and oswald, 1994 ; stack and eshleman, 1998 ; layard, 2005 ; clark, 2006 ; blanchflower and oswald, 2008 ; dolan., 2008 ; oswald and powdthavee, 2008 ; diener., 2009b). there is, therefore, a pressing need to analyse the new social survey data with multivariate regression and modelling techniques (where all the different socio - demographic characteristics are taken together and controlled for) in order to shed more light on the correlates of swb in the uk. although officials have not undertaken this work themselves, they are actively encouraging the research community to do so. table 2.average (mean) ratings for the four overall subjective monitoring questions by personal characteristics (sex, age, self - reported health and long standing illness or disability : great britain, adults aged 16 and over)life satisfactionworthwhilehappyanxioussexmen7.37.57.33.3women7.57.87.53.6age16197.87.87.83.720247.47.77.33.325297.17.47.23.630347.47.57.43.335397.07.57.13.740447.37.67.23.745497.27.57.23.350547.47.77.23.555647.47.77.43.665747.97.97.83.075 or over7.77.47.63.1healthvery good7.98.17.93.1good7.47.67.43.4fair6.87.06.74.0bad5.56.05.55.1very bad4.24.64.85.0illness / disabilityyes7.07.27.03.8no7.67.87.63.3source : reference tables for investigation into subjective well - being data from the ons opinions survey (ons, n.d.). average (mean) ratings for the four overall subjective monitoring questions by personal characteristics (sex, age, self - reported health and long standing illness or disability : great britain, adults aged 16 and over) source : reference tables for investigation into subjective well - being data from the ons opinions survey (ons, n.d.). logistic regression, in spss (version 19.0), was used to identify the social determinants of swb in the national population. we use the four global measures of swb discussed above (with the relevant ons variable codes shown in table 3), and we draw on the range of socio - demographic factors captured by the ons survey (table 4) to examine the risk and relative odds of low swb in the british population (ons survey described in the appendix below). table 3.overall measures of subjective wellbeingvariablevariable labelmonitoring questionmcz_1life satisfactionoverall, how satisfied are you with your life nowadays?mcz_2worthwhileoverall, to what extent do you feel the things you do in your life are worthwhile?mcz_3happyoverall, how happy did you feel yesterday?mcz_4anxiousoverall, how anxious did you feel yesterday?source : ons (2011c, 2012). table 4.dependant variables in the modelvariabledescriptionspecification in the studyagexageage may help to explain wellbeing in the british population. here age is recoded into six groups.rsexsex/gendersex/gender may help to explain wellbeing in the british population (male / female).ethnicityto which of these groups do you belong?ethnicity may help to explain wellbeing. responses to this question are recoded into two groups : white and black and minority ethnic (bme).dvilo4adv for ilo in employment (four categories)being in work may help to explain wellbeing, here we have four categories : in employment (exc. unpaid family workers) unpaid family workers unemployed (ilo definition) economically inactive.sumgrossgross annual incomeincome may help to explain wellbeing. responses to this question are recoded into income quintiles.ten1housing tenurethree groups : home - owner (including those with a mortgage) private renter social housing (including housing association accommodation or local authority housing).defact1de facto marital status (grouped)household composition may help to explain wellbeing. responses to this question are recoded into two groups : couple (includes married, cohabiting, civil partner) single (living alone, inc. divorced, separated, widowed).qhealthhow is your health in general?self - reported health may help to explain wellbeing. bad and very bad.lsillhave any long - standing illness, disability or infirmity?long - standing illness and disability may help to explain levels of wellbeing in the british population. no.highed4what is the highest level of qualification?education measured by educational attainment may help to explain wellbeing in the british population. there are three categories : degree or equivalent, below degree level, none (no qualifications).nsecac3national statistics socio - economic classification (ns - sec)social class and socio - economic position may help to explain patterns of wellbeing (we use the standard ns - sec 8 classification).goragovernment office regionwellbeing in britain may vary by geography and region of residence.source : ons (2011c, 2012). dependant variables in the model source : ons (2011c, 2012). logistic regression is a statistical technique that belongs to the theoretical framework of the general linear models (glm), described by dobson (2001), and is ideally suited to situations where a continuous response variable, such as swb, has been categorised as a dichotomy using binary coding (hosmer and lemeshow, 2000). for the analysis therefore, we have created new binary variables on each measure of wellbeing : for unhappiness, respondents who did not answer the survey questions on wellbeing are not included in this study. first, our bivariate logit models establish the relative odds of wellbeing, along the different dimensions, by the range of individual and household characteristics, but without taking account of any of the other variables (the results are shown in table 5). a bivariate model is a fairly simple one that shows the relationship between two variables, although many predictive factors are likely to be interrelated there are often clear links between age, health and income for instance. importantly, the relative odds of reporting or predicting high or low levels of swb independent of other variables can be calculated using a multivariate model (independent here means after taking account of all of the other demographic and socio - economic variables in the model). much of the discussion below focuses on the findings from the multivariate analysis shown in table 6, with cross - referencing to the bivariate findings in order to help understand some of the complexity surrounding the social determinants of swb. finally, we examine for covariance and interactions between variables in the main effects multivariate model (results are shown in tables 6 and 7). table 5.the relative odds of wellbeing (bivariate model)unhappydissatisfiedunfulfilledanxious(i)(ii)(iii)(iv)sex / gendermale1.001.001.001.00female1.091.34 1.450.82ethnicitywhite1.001.001.001.00bme1.591.661.53 1.51age16241.001.001.001.0025441.181.441.061.1945541.271.541.011.0355640.971.220.821.2265740.820.890.860.9075 + 0.881.281.75 0.76healthvery good1.001.001.001.00good1.621.902.531.19fair2.973.826.501.79poor8.1413.119.72.74very poor11.225.133.92.66disabilityno rated disability1.001.001.001.00reported disability1.952.292.961.44educationdegree1.001.001.001.00below degree1.541.251.61 1.03no formal qualifications1.921.742.531.08labour force statusin employment1.001.001.001.00unemployed2.133.933.581.30economically inactive1.201.562.401.11socio - economic positionmanagerial / professional1.001.001.001.00intermediate0.981.341.020.91manual workers1.462.032.140.99income quintiletop1.001.001.001.00second0.921.441.280.83middle1.052.172.221.03fourth1.43 2.843.701.31bottom1.42 3.303.171.28household compositioncouple1.001.001.001.00single person1.762.723.331.06housing tenurehome owner1.001.001.001.00private rental1.911.812.261.15social housing2.353.124.011.04region of residencenorth east1.001.001.001.00north west0.740.750.790.67yorkshire & the humber0.790.881.020.59east midlands0.670.610.740.68west midlands0.931.010.790.77east of england0.730.770.690.54london1.141.741.610.87south east0.610.720.550.72south west0.51 0.700.630.77wales0.870.921.290.67scotland0.831.150.850.66notes : significance levels : < 0.05 ; < 0.01 ; < 0.001. table 6.the relative odds of wellbeing (multivariate model)unhappydissatisfiedunfulfilledanxious(i)(ii)(iii)(iv)sex / genderfemale1.001.001.001.00men1.051.301.630.82ethnicitywhite1.001.001.001.00bme1.59 1.181.181.24age16241.001.001.001.0025441.182.13 1.291.1845541.132.37 1.071.0155640.771.110.641.0065740.560.980.500.7375 + 0.44 1.070.990.74healthvery good1.001.001.001.00good1.43 2.082.031.25fair3.135.666.701.84poor7.9018.119.62.73very poor10.353.829.83.28disabilityno rated disability1.001.001.001.00reported disability1.070.951.101.18educationdegree1.001.001.001.00below degree1.651.151.621.21no formal qualifications1.880.941.221.20labour force statusin employment1.001.001.001.00unemployed1.412.611.651.15economically inactive0.930.731.080.92socio - economic positionmanagerial / professional1.001.001.001.00intermediate0.801.220.740.82manual workers0.991.44 1.240.87income quintiletop1.001.001.001.00second0.831.290.980.83middle0.701.451.151.00fourth0.811.431.471.20bottom0.912.17 1.271.16household compositioncouple1.001.001.001.00single person1.772.792.640.97housing tenurehome owner1.001.001.001.00private rental1.49 1.061.251.03social housing1.041.171.320.80region of residencenorth east1.001.001.001.00north west0.710.700.810.73yorkshire & the humber0.660.941.150.64east midlands0.590.740.780.70west midlands0.750.780.620.67east of england0.52 0.720.700.59london0.761.802.130.77south east0.580.950.770.76south west0.43 0.930.710.86wales0.840.711.340.82scotland0.791.170.810.80notes : significance levels : < 0.05 ; < 0.01 ; < 0.001. table 7.the relative odds of wellbeing (significant interactions in the main effects model)unhappydissatisfiedunfulfilledanxious(i)(ii)(iii)(iv)incomehealthsecond quintile good healthmiddle quintile good health1.79fourth quintile good healthbottom quintile good health1.52second quintile fair health2.14middle quintile fair health2.32fourth quintile fair health2.274.004.812.24bottom quintile fair health2.224.892.79 2.20second quintile poor health2.24middle quintile poor health18.403.311.89fourth quintile poor health4.747.296.493.01bottom quintile poor health4.5014.5410.782.25second quintile very poor health7.5014.267.56 5.60middle quintile very poor health10.1221.3622.703.38fourth quintile very poor health12.9861.8232.455.63bottom quintile very poor health7.0643.6428.057.42incomeemploymentsecond quintile unemployedthird quintile unemployedfourth quintile unemployed2.64 4.29bottom quintile unemployed3.701.95disabilityhealthdisability good health2.00disability fair health2.122.963.091.88disability poor health5.8010.1211.092.63disability very poor health7.7929.0516.423.22healthemploymentunemployed good healthunemployed fair healthunemployed poor health3.734.536.311.88unemployed very poor health4.8614.4610.132.37disabilityhousehold compositiondisability single person1.621.851.84ethnicityemploymentwhite unemployment2.17bme unemployment3.74 4.72socio - economic positionhealthintermediate good healthintermediate fair health2.322.47intermediate poor health4.087.336.222.10intermediate very poor health5.0913.348.71manual good healthmanual fair health2.273.483.011.47manual poor health5.4310.646.331.94manual very poor health11.2533.0011.462.13socio - economic positioneducationintermediate below degreeintermediate no formal qualifications2.44manual below degreemanual no formal qualifications1.46 1.59 1.76household compositiontenuresingle person private rental2.522.182.48single person social housing1.811.842.45significance levels : < 0.05 ; < 0.01 ; < 0.001. the relative odds of wellbeing (bivariate model) notes : significance levels : < 0.05 ; < 0.01 ; < 0.001. the relative odds of wellbeing (multivariate model) notes : significance levels : < 0.05 ; < 0.01 ; < 0.001. the relative odds of wellbeing (significant interactions in the main effects model) significance levels : < 0.05 ; < 0.01 ; < 0.001. in the statistical models, those who report being satisfied with life, not suffering with anxiety or leading worthwhile lives (being the majority in each case) form the base in each model. anxious or leading an unfulfilled life by the range of socio - demographic factors shown in table 4., with the relative odds reflecting the odds of one particular category compared to the reference. these odds ratios show the strength and the direction of the predictors asterisks indicate the level of significance and the base case is always 1.00. all study calculations are weighted (see appendix) to correct for non / differential response rates, in order to ensure study estimates relate to the national picture (crockett, 2006). a first step in the analysis was to examine the survey data relating to swb in the british population. overall, the survey results suggest that wellbeing in the national population is largely positive, not negative, and this is to be expected (deaton, 2008). 92.4 per cent compared to just 7.6 per cent who are not. according to the survey fulfilled. anxiety is more prevalent however : over a third of people (37.4 per cent) report feeling anxious about life when surveyed, leaving 62.6 per with little or no such anxiety. (column (i)), we observe that age, ethnicity, health, education, household composition and housing tenure continue to have an impact on happiness in great britain, even after controlling for all other factors in the model, as does geographical region. happiness are moderated by other socio - demographic characteristics in the model, notably by health status, according to the strong interactions with income (observed in table 7), and employment status to a lesser degree. gender, social class and labour - force status do not appear to have much of an impact on happiness after controlling for everything else ; neither does disability. unhappiness in the bivariate model, but has an independent effect in the multivariate analysis. in table 5, for instance, the odds of adults who report a disability being unhappy are twice those of non - disabled adults. however, the effects of disability are significantly diluted or covered by the inclusion of other variables in the model (suggesting evidence of multicollinearity here), particularly with the more global assessment of health status, itself a powerful predictor of swb across all four dimensions (columns (i)(iv)). happiness. other things being equal, the odds of people in very poor health being unhappy are ten times those of people in very good health (column (i)) ; the odds for those in poor health are eight times greater and three times greater for those who report their health is fair. the findings for socio - economic status are more curious ; we find manual workers are significantly more likely to be unhappy without controls, whereas the social class effects on happiness are clearly moderated with the controls in place. the results also show an age effect : people in the oldest age group (seventy - five - plus) are significantly more likely to be belonging to a black and minority ethnic (bme) group also significantly increases the relative odds of being there are also important interactions between ethnicity and other variables in the multivariable model, which are discussed in the section on interactions below. the relative odds of being unhappy increase significantly with lower levels of educational achievement. for example, the relative odds of reporting unhappiness for those with educational attainment below the level of university degree is two - thirds greater than those with a university degree. those with no formal qualifications display unhappy are 50 per cent greater than those of a home - owner (which includes people buying their own home with a mortgage). furthermore, there appears to be regional variation in levels of happiest place to live, being consistent with other surveys of swb (anand., 2009). the region is unremarkable on traditional economic indicators however, for example, gdp or gva (ons, 2011d), thus providing further caution against taking simple economic statistics as measures of social progress and wellbeing, as harvie. (2008) argue. on the life satisfaction measure (column (ii)) of self - assessed wellbeing manual workers and those at the bottom of the income distribution all report higher levels of dissatisfaction with their lives, for instance, are more than 40 per cent greater than representatives from the managerial and professional classes. we also see in table 6 that the odds of respondents in the lowest income quintile reporting dissatisfaction are more than twice those of respondents in the top income quintile. life satisfaction. as expected, during the period of middle age (twenty - five to fifty - four) there is increased dissatisfaction with life. although this familiar u - shape to wellbeing across the life - course is not fully understood, clearly this is a time of struggle for most adults, as they start to build a family and buy a home (blanchflower and oswald, 2008). other things being equal, the odds of single people living alone reporting to be dissatisfied are now three times greater than those of people living together as a couple. ethnicity appears to be less decisive on this particular dimension of wellbeing, at least after controlling for all the other variables in the multivariate analysis. people from bme backgrounds appear to experience the same or similar levels of life satisfaction as the majority white british population. however, since we observe significant levels of dissatisfaction in the bivariate analysis we may wish to question the adjustments in the multivariate model. at times it may be problematic to give the appearance of having adjusted for, and thereby having removed, any socio - economic differences in the statistical model while much of it remains in real life (karlsen., 2012). dissatisfied with life in the bivariate analysis, and yet disability is no longer significant when all of the other variables are controlled for in the model. again this suggests that it is the social barriers and some of the observed health effects that impinge upon those who report an impairment (roulstone and prideaux, 2012). in other words, it is the circumstances of living with a disability that matter rather than simply the notion or label of disability itself. dissatisfied for a disabled person living alone are nearly twice those of a disabled person living in a couple (table 7). the independent health effects observed are even more pronounced than those found on the measure of dissatisfaction amongst people who claim their health is very poor are now over fifty times greater than those of people who report that their health is very good (table 6). while the odds of unhappiness for those in poor health are eighteen times greater and six times greater for those who claim only fair health. happiness but not so for life satisfaction. there was little or no meaningful variation according to educational attainment. conversely, while labour market participation had little or no effect on happiness, employment now seemingly alters perceptions of life satisfaction. the odds of an unemployed person being dissatisfied with life are more than two - and - a - half times those of someone in work. interestingly, someone who is economically inactive, having retired from paid work or being a full - time student, is much more likely to be satisfied with life. unlike the findings relating to happiness, we observe no meaningful difference in the global assessments of life satisfaction by geographical region. in other words, life satisfaction appears to be fairly evenly distributed across the regions of great britain. although research has yet to identify the precise constituents that add meaning to our lives, and help make them worth living, we can, nonetheless, consider some of the relative socio - demographics associated with the life fulfilled (column (iii)). in many ways, the multivariate results for worthwhile lives (a measure of women, for instance, are significantly more likely to report a stronger sense of purpose and meaning to their lives than men. this is interesting, not least because we found no significant difference by sex / gender on the happiness and global life satisfaction measures of swb. unfulfilled lives amongst people who self - report very poor health, for example, are now thirty times greater than those in very good health (table 6). while the odds for those living in poor health are now twenty times greater, and over six times greater for those who report their health is fair. with health status and all other variables controlled for in the model, there was no significant difference on this measure of swb between those who report a disability and those who do not. again, this suggests that many of the issues and disadvantages experienced by people with a disability are located within the social and economic circumstances of everyday life (roulstone and prideaux, 2012). living alone is consistently significant, as a person with a disability who lives alone displays nearly twice the odds of leading an unfulfilled life compared with a disabled person who does not live alone (table 7) ; as might be expected, good health is also highly significant. worthwhile which are three times greater than those of single people living alone. housing tenure, however, was not a good predictor on this measure of swb. age, ethnicity, education, income and social class appeared to play little or no significant role in shaping, geographical region currently plays no major role in shaping the extent to which people feel their lives are fulfilled and worthwhile. although women lead more meaningful lives, they also report significantly more general age and ethnicity are not especially important on this particular dimension of swb. in the multivariate analysis, people with bme backgrounds experience the same or similar levels of stress as people in the white british population. there is the now familiar gradient with health status, but the observed differences here are not as pronounced as we found using the other measures of swb. for instance, the odds of people in poor health being this difference is highly significant, but the order of magnitude on the measures of life satisfaction and worthwhile life was far greater. income, socio - economic position and labour force status do not appear to impact on anxiety after controlling for other factors ; neither does disability. the latter finding is very much in keeping with the other results for swb in table 6. income and disability were significant predictors of anxiety in the bivariate analysis, but the effects of income and disability continue to be moderated by the other socio - demographic variables including health status in the multivariate model. again, we find strong interaction effects between income and self - rated health, along with disability and self - rated health (table 7). the study findings also suggest that people living in yorkshire and in the east of england are significantly less anxious when all other factors in the model are controlled for. the results suggest that there is some correlation between some of the variables in the multivariate analysis. the effects of disability, for example, are significantly diluted or covered by the inclusion of other variables in the multivariate model shown in table 6, particularly the more global assessment of health status ; this is a powerful predictor of swb across all four dimensions (columns (i)(iv)). in the interaction models (table 7), we observe strong interaction effects between states of health and disability, showing the impact of declining health on swb. dissatisfied amongst adults with a disability who are in very poor health, for example, are now about thirty times those of a disabled adult in very good health. the interaction effects between disability and health illustrate the importance of good health for positive wellbeing. we also observe strong interaction effects between disability and household composition (table 7). an adult with a disability living alone is significantly more likely to report low levels of swb compared to an adult with a disability living with another person as a couple. in table 7, we find strong effects between income and self - rated health. as health declines, the odds of reporting low swb increase substantially regardless of income ; however, the effects are much more dramatic at the lower end of the income distribution (from the lower middle - income quintile to the bottom quintile). in addition, the evidence suggests that income makes more of a difference to general wellbeing during the earlier stages of adult life (twenty - five to forty - four), and in middle age (forty - five to fifty - four) on the satisfaction measure of swb. income appears to play a less significant role in shaping wellbeing in later life (seventy - five - plus), particularly for the measures of happiness and stress free living. younger adults living alone are more likely to report significantly low levels of swb compared to older adults living alone. we also observe significant interactions between age and health as might be expected ; adults aged twenty - five to fifty - four in poor health (including the very poor health category) tend to be significantly more similarly, levels of dissatisfaction and unfulfillment also tail off with age. table 8.the relative odds of wellbeing (significant interactions in the main effects model)unhappydissatisfiedunfulfilledanxious(i)(ii)(iii)(iv)agehealth2544 good health2544 fair health3.236.302544 poor health18.694.7839.556.152544 very poor health17.23 33.8225.984554 good health4554 fair health2.394.274554 poor health5.8823.3222.224554 very poor health12.04 47.887.325564 good health5564 fair health2.742.43 3.525564 poor health10.664.275564 very poor health4.60 34.5729.456.81 6574 good health6574 fair health5.376574 poor health4.216574 very poor health4.2228.9021.4775 + good health75 + fair health3.6575 + poor health3.05 8.374.1675 + very poor health8.4011.83 13.57agehousehold composition2544 single person2.023.223.334554 single person4.042.775564 single person1.72 2.542.296574 single person75 + single person2.03ageincome2544 second quintile2544 middle quintile2.012544 fourth quintile1.49 2544 bottom quintile4.822.214554 second quintile4554 middle quintile0.69 4554 fourth quintile2.564554 bottom quintile2.685564 second quintile0.57 5564 middle quintile0.235564 fourth quintile5564 bottom quintile6574 second quintile6574 middle quintile6574 fourth quintile6574 bottom quintile75 + second quintile75 + middle quintile75 + fourth quintile0.310.56 75 + bottom quintilesignificance levels : < 0.05 ; < 0.01 ; < 0.001. the relative odds of wellbeing (significant interactions in the main effects model) significance levels : < 0.05 ; < 0.01 ; < 0.001. there are also important interactions between ethnicity and other variables in the multivariable model. unhappy are nearly four times greater than those of the majority white british population, and their odds of reporting unemployed adults reporting poor health have significantly lower levels of swb compared to adults with poor health who are in work. employment does appear to confer some benefits on those who are able to work, even if their health is not good (table 7). manual workers in poor health (including the very poor health category) are shown to have the lowest levels of wellbeing, alongside manual workers without any formal qualifications. household composition also matters ; people living alone in rented accommodation or social housing face twice the odds of being unhappy, dissatisfied and feeling it is now widely accepted that the measurement of swb is central to the development of social policy. measures that capture personal experience, moods, psychological functioning and people 's cognitive assessments about how their life is going overall when aggregated at the national population level elucidate an important component of the wellbeing concept that can be applied to social policy (graham, 2011). the emerging survey data on swb in the uk, however, is currently underdeveloped for social policy purposes. in this article, we have considered the application of the survey data for understanding the social determinants of swb, and here we reflect on how the new insights may be used to inform the development of social policy. a number of findings are particularly striking, and deserve comment given the direction of current policy in the uk. work is increasingly recognised as the best form of welfare in twenty - first century capitalist society (dwp, 2010), not only because work usually pays better than welfare, but because work helps to promote wellbeing, as we observe here : the odds of dissatisfaction amongst adults not in work are nearly three times greater than those in work, according to the survey data. to this end, the uk 's new welfare - to - work policies, including the work programme and the new means - tested benefit universal credit seek to ensure welfare that works ; a life in work rather than a life on benefits (welfare reform act, 2012). consequently, labour market conditionality now applies to most sections of the adult working age population and the principles at stake here appear to be attracting support amongst the british public (park., 2011). in many ways and from the perspective of swb the policy aim is laudable ; there are obvious benefits and gains if more out of work adults are able to find decent, meaningful jobs. however, there are growing concerns over the level of state regulation in the name of labour market activation, including some of the present structural concerns over the lack of employment opportunities on offer, as well as the high cost of childcare faced by many working parents. the limits of these policies are now being tested by some of the most vulnerable sections of british society, who maintain that their needs are not being respected under the new regulatory regime. activation policy, claim that taking a paid job is not appropriate in their circumstances (rafferty and wiggan, 2011). instead, they express a strong preference for caring for their own children themselves. likewise, the government policy to encourage and assist disabled people off benefits and in to paid work is surrounded with controversy for ignoring the needs of disabled people, and societal barriers to their full and equal labour market participation (mccartney, 2012 ; patrick, 2012). paid work will not be appropriate in every circumstance and not all adults will benefit from being satisfaction for disabled adults, in stark contrast with the findings for the general british population. many older adults in retirement report relatively high levels of swb ; empirical research continues to show most lead socially active and productive lives. the uk 's active ageing strategy, therefore, needs to support local big society initiatives, and thus extend beyond the rather narrow focus on employment opportunities (deeming, 2009). whether increasing income raises the experience of wellbeing is a matter of major debate (diener., 2009b). in this study, we found little evidence to suggest that income makes an independent contribution to swb across our four dimensions. however, we should recognise the need for more sophisticated modelling techniques that include both individual and societal factors to probe this complex relationship (deeming and hayes, 2012). it is noticeable, and also worrying, that there has been a distinct shift in policy away from faith in income and material recourses to solve some of the complex and deep - rooted social problems we see in the uk (e.g., field, 2010) ; and while we wait for research to uncover more truths, we should not lose sight of the powerful effect that income and wealth has on important social outcomes (hills. fulfilled lives than men, a finding that is very much in tune with some of the emerging international evidence (oecd, 2012), it is also very apparent that women are more this is as a result of combining employment and family care, as lewis (2009) argues. one - size - fits - all regulatory framework being imposed upon them by welfare - to - work. ensuring that families, especially single parent families, are able to both support themselves and to care for their dependants without material disadvantage continues to be a major challenge for social policy in the uk. the issues raised here are symptomatic of more general shifts in the balance of work and family life, with ever greater family diversity and changes in the labour market. ageing populations and changing family structures present new challenges. policymakers have been slow to respond to the new social risks of modern societies in the twenty - first century as taylor - gooby (2004) argues. generally speaking, living alone is not good for wellbeing as the study findings and a growing body of research evidence indicate (stack and eshleman, 1998). solo living often results in isolation from the privileges and obligations of cohabited (including married) life. the ties of cohabitation help to establish a basic sense of belonging and security ; swb is clearly affected where such ties are lacking. perhaps a growing area of concern for the policy of wellbeing is the growing numbers who live alone in britain : a trend which shows little sign of abating (chandler., 2004). although the ageing population has contributed to this demographic, solo living is a growing trend across all age groups in the adult population. individualisation means people are inextricably linked to the institutions of the labour market and welfare state, while traditional forms of family and community life lose more and more of their meaning. the new institutions and social structures increasingly regulate life, with demands and entitlements that are not wholly addressing the needs of individuals, and nor are they addressing the needs of family variety in the twenty - first century. policymakers face pressing issues over intergenerational equity, as the study findings indicate wellbeing is not evenly distributed between the generations (willetts, 2010). structural components of ethnic disadvantage persist, despite various employment initiatives and legislation (virk, 2012). the results suggest that swb among unemployed people in bme groups are a particular concern. the interplay of factors at stake here is likely to be complex as berthoud (2000) suggests, but will include known factors such as overt and hidden discrimination, expectations, stereotypes, alienation, family and economic structures. education helps to promote wellbeing, and is well - recognised as a cultivating force within society (nussbaum, 1997). however, the recent reforms to higher education in england are, according to some critics such as barr (2012), a retrograde step that is likely to undermine the quality of education and the goal of widening participation. students from poorer backgrounds already experience a range of structural disadvantages in the higher - education system, relating to economic, social, and cultural capital (lehmann, 2009). expensive loans are likely to further impede access and weaken participation, stifling the prospects of social mobility and increased wellbeing for young people from poorer working - class backgrounds. we find that people living in poor health which often means coping with a longstanding illness are amongst the most vulnerable members of british society, reporting the lowest levels of wellbeing. there are clear links between age and health in the data as expected, but poor health also interacts with other forms of social and material circumstance, such as unemployment, low income, low social class and self - rated disability (clark and oswald, 1994). new strategies demand that individuals take greater responsibility for their own agency and wellness. power and responsibility is being devolved to the local level ; wellness services in each locality are to be held accountable for the wellbeing of their local populations (health and social care act, 2012). the danger of this fiercely consumerist and individualist self - directed approach to policy is the increasing fragmentation and marketisation of the social and human services ; particularly evident in the new public health paradigm (bambra., 2011), social care policy (glendinning, 2012) and housing policy (lund, 2011). better government regulation of the private rental sector is urgently required to promote health and wellbeing, although this is not a priority for the coalition government. nearly 40 per cent of privately rented homes fail the government 's decent homes standard, and people in this sector express high levels of. moreover, many vulnerable families in britain are being funnelled into the bottom end of this market, resulting from the caps on housing benefit imposed by the new system of universal credit (harding, 2011). the policy turn to individual consumerism and the market, with the desire to devolve responsibility from whitehall, and arguably shift blame, threatens the very foundations of human health and wellbeing in the uk at a time when the influence of the wider social context is being ignored. there is an urgent need to emphasise the social, given the direction of current policy in britain. research into wellbeing, both the subjective (swb) components and the substantive (i.e., holistically understood) aspects of human flourishing belong to the core business of social policy (dean, 2012). here we have focused on swb and the emerging mnw programme that offers much potential for the development of social policy, informed by the rigorous analysis of survey data within the social scientific tradition (byrne, 2011). there are, of course, a number of cautions and limitations attached to the study findings. correlations in this field should not be treated as evidence of one - way causation from factors to the cross - sectional survey data used here is limited when it comes to studying changes in swb over time and/or across the lifecourse. longitudinal studies, cohorts or panel surveys are required for modelling and interpreting cross - temporal comparisons that shed light on whether the same population has become happier (davies, 1994). researchers may wish to consider a radically different approach to the interpretation of this survey data using a set - theoretic method for example. finally, and more conceptually, we are a long way off from a world in which emotional fulfilment replaces ideas about maximising economic growth, and although social policy is making progress, there would be grave dangers in any single - minded pursuit of this (burchardt, 2006). the value of social policy is that it recognises the plurality of human ends and needs (not just ideas relating to happiness or swb), and as such it invokes our social values and principles for thinking about resources, material circumstances, and their distributions. in terms of future work, there is a more general need to examine more closely the relationship between swb and mental health issues (beaumont and thomas, 2012). further work should also consider disparities in swb within and between minority ethnic groups within society (burton., 2010 ; platt, 2011). finally, the four overall swb monitoring questions now included in ons social surveys are designed to collect different types of information from respondents about their own wellbeing ; ons argue that the four swb constructs and measures can and do stand alone, and while there is a literature to support this thinking, future work might also test some of these assumptions since these measures and constructs are now widely used internationally.	the idea that the happiness and wellbeing of individuals should shape government policy has been around since the enlightenment ; today such thinking has growing practical policy relevance as governments around the world survey their populations in an effort to design social policies that promote wellbeing. in this article, we consider the social determinants of subjective wellbeing in the uk and draw lessons for social policy. survey data are taken from the measuring national wellbeing programme launched by the uk 's office for national statistics in 2010. for the empirical strategy, we develop bivariate and multivariate logistic regression models, as well as testing for interaction effects in the data. the findings show that wellbeing is not evenly distributed within the uk. socio - demographic characteristics such as age, gender, ethnicity, employment, household composition and tenure all matter, as does health status. influencing population wellbeing is inherently complex, though, that said, there is a clear need to place greater emphasis on the social, given the direction of current policy.
comorbidities are defined as diseases that are concomitant to the disease under study.1 when survival in medical and epidemiological research is studied, it is important to consider the effect of comorbidity, as it could be a potential confounder or an effect modifier for other prognostic factors.26 the charlson comorbidity index (cci) and the elixhauser method are the most commonly used methods to assess comorbidity.7,8 the cci is used more often, is less complex than the elixhauser method, and was therefore chosen as the reference index in the present study. the cci was originally developed based on data from all patients admitted to the new york hospital cornell medical center during a 1-month period in 1984, with the objective of predicting 1-year mortality. the risk of death associated with each of the 19 predefined diseases included in the cci was expressed as weights with values of 1, 2, 3, or 6. summing the weights for all contributing diseases gives the cci score for each patient. the cci was validated using an external cohort consisting of 685 breast cancer patients receiving their first treatment in 19621969 at yale new haven hospital.7 the original cci has been modified and evaluated by many authors.913 quan developed coding algorithms for constructing the cci based on the codes of the tenth revision of the international classification of diseases (icd-10).14 sundararajan assessed that the version of the cci created by quan outperformed all the other versions they considered, though all of the icd-10 versions of the charlson algorithm performed satisfactorily.15 however, a persistent challenge for researchers who wish to use the cci is that the data set of interest may not contain sufficient medical information to assess all the diseases included in the index. these are based on hospital administrative databases, from which data are often readily available. due to better treatment and technological improvements, a patient s risk of death has reduced since the cci was established in the 1980s, and the index has been updated accordingly. bottle and aylin12 and quan both updated the cci.11 bottle and aylin used national data from the uk and studied in - hospital mortality, while quan used regional data from the calgary health region of canada to study 1-year case fatality. however, to our knowledge, no previous study has combined the use of national patient - register data and death within 1 year as the study end point. the aim of the present study was to evaluate the importance of including a comorbidity measure into analyses when predicting 1-year mortality using data from the norwegian patient register (npr). a second aim was to construct a modified version of the cci that can be used with patient - register data (the patient register index, pri), and compare the predictive ability of the cci and the pri, as well as the confounding effect of each index on age. an additional aim was to describe the pattern of diseases included in the cci by age and sex, based on data from all hospitals in norway registered in the npr. the npr is a national health register covering all sectors of the specialized health care services. reporting to npr is mandatory, and the register includes data on all patients treated in norwegian government - funded hospitals. this enables researchers and policy makers to follow the disease trajectory of patients between sectors and hospitals. also, alignment of data and validation with other national health registries is made feasible. the study sample was selected from the npr data reported from all hospitals, which consisted of three main data sources for statistics. the first source was visits for medical treatment for in- and outpatients at publicly financed hospitals.. the government purchases medical treatment from private hospitals and specialists practices as a supplement to services at the public hospitals. however, when a patient is transferred between wards at the same hospital, the record is aggregated. each episode of national hospital data contains one or more diagnoses, coded according to the icd-10. to provide information on the total disease history of the patient, the personal identification number was used to link episodes of treatment registered at different hospitals, sectors, and years. initially, all patients in the npr registered with a hospital visit in 2010 or 2011 were identified, constituting 15,214,796 visits. the first group was outpatients at private specialist practices in 2010 and 2011 (3,359,618 visits). the reason for incomplete reporting in this group was mainly related to technical limitations in their information - technology systems. to avoid any selection bias however, we decided to keep outpatient visits with surgical procedure codes, because this activity mainly is performed at publicly financed hospitals or publicly financed private hospitals. the second group of visits excluded due to incomplete reporting were data on newborn babies (139,196 visits), since a large proportion had not yet received a personal identification number at the time of registration. the third group was any other patient visits that were lacking a correctly reported personal identification number (49,249 visits). after these exclusions, the data were aggregated, resulting in 1,534,942 eligible visits, corresponding to 1,113,341 unique patients. the first hospital visit during the study period was defined as the patient s index visit. the end of follow - up was defined as the date of death if the patient died within 1 year of the index visit, or the date 1 year after the index visit (censoring date). sex, age category, and icd-10 codes (truncated to three digits for the index visit) were obtained from the npr. to avoid the possibility of tracing back to individual patients duration in days from the index visit to end of follow - up, vital status, and history of the diseases (dichotomous variables) included in the cci that were listed up to 1 year prior to the index visit were obtained from the npr. the npr routinely obtains information on vital status and date of death from the national population register.16 the data file obtained from the npr was anonymized, and thus no requirement for regulatory ethical approval in norway was needed. the original cci included 19 diseases, but for the purposes of the present study, leukemia and lymphoma were included in the disease category any malignancy, as done by other authors.911 these 17 cci diseases were identified by the icd-10 coding defined by quan in 2005.14 a primary diagnostic code is the code for the main medical condition causing the admission. secondary diagnostic codes are codes for diseases that exist at the same time as the primary disease, or diseases that develop and need examination or treatment during the admission. the pri weights were estimated using a cox regression model of time from index visit to death with follow - up censored at 1 year, adjusted for sex, 5-year age categories (04 years an algebraically correct method was used to define new weights for the cci diseases by summing the regression coefficients, not the hazard ratios (hrs).17 the pri was constructed by multiplying the regression coefficients by a scaling constant k, then rounding it to the nearest integer, and finally summing it over all the cci diseases. the scaling constant k was chosen such that the maximum weight for a specific disease in the pri became the same integer as the maximum weight in the cci, and thus k was set to 2.3. both the construction and comparison of the pri and the cci were done through an internal tenfold cross - validation procedure.18 ten subsets of the data were randomly defined, and nine of them were used to develop the pri, with the last subset used to run the comparison. this was done for all ten subsets, and the results are given as the mean values from the ten runs. to predict 1-year mortality, three logistic regression models were fitted, one including sex and age (base model), one including sex, age, and the cci (cci model), and one model including sex, age, and the pri (pri model). model fit was compared using several measures of global fit. the bayesian information criterion (bic) is a likelihood - based measure, and for model fit, the lower the bic - value, the better the fit.19 likelihood ratio chi - square statistics were also used to test model fit. mcfadden s r is a measure of improvement in fit over the intercept model.20 the brier score was used to directly compare the observed outcomes with the predicted probabilities.19 the c - statistic, which is a summary of a model s ability to discriminate between those who do and those who do not experience the outcome, was calculated for each model.21 this measure is the most commonly used in the medical and epidemiological literature when comparing different comorbidity indices.11,13,15,22,23 the c - statistic varies from 0.5, which indicates that the discrimination is due to chance alone, to 1, which indicates perfect discrimination. the general classification of discrimination is acceptable (c [0.7, 0.8 ]), excellent (c [0.8, 0.9 ]), and outstanding (c [0.9, 1]).24 to assess which index changed the predicted value most when added to the base model, movement from the estimated probabilities of the outcome were compared. this was done using the net reclassification improvement (nri), which is defined as the difference in proportions moving up or down in risk among patients who died and those who survived. we also used the integrated discrimination improvement (idi), which measures the difference in the mean predicted probabilities between those who died and those who did not in the cci and pri models compared to the base model.25,26 we also calculated how much the probabilities changed on average when a comorbidity index was included in the model : this quantifies the effect of introducing a comorbidity index into a model. to compare the importance of including age, sex, and a comorbidity index in the model, p^ was calculated with and without each of the variables in a subanalysis with patients aged over 50 years who were not admitted to hospital due to any of the 17 cci diseases at the index visit. in the description of the disease pattern, the patient group was age - standardized according to the norwegian standard population in 2011.27 all analyses were done using stata 13.28 the npr is a national health register covering all sectors of the specialized health care services. reporting to npr is mandatory, and the register includes data on all patients treated in norwegian government - funded hospitals. this enables researchers and policy makers to follow the disease trajectory of patients between sectors and hospitals. also, alignment of data and validation with other national health registries is made feasible. the study sample was selected from the npr data reported from all hospitals, which consisted of three main data sources for statistics. the first source was visits for medical treatment for in- and outpatients at publicly financed hospitals.. the government purchases medical treatment from private hospitals and specialists practices as a supplement to services at the public hospitals. however, when a patient is transferred between wards at the same hospital, the record is aggregated. each episode of national hospital data contains one or more diagnoses, coded according to the icd-10. to provide information on the total disease history of the patient, the personal identification number was used to link episodes of treatment registered at different hospitals, sectors, and years. initially, all patients in the npr registered with a hospital visit in 2010 or 2011 were identified, constituting 15,214,796 visits. the first group was outpatients at private specialist practices in 2010 and 2011 (3,359,618 visits). the reason for incomplete reporting in this group was mainly related to technical limitations in their information - technology systems. to avoid any selection bias however, we decided to keep outpatient visits with surgical procedure codes, because this activity mainly is performed at publicly financed hospitals or publicly financed private hospitals. the second group of visits excluded due to incomplete reporting were data on newborn babies (139,196 visits), since a large proportion had not yet received a personal identification number at the time of registration. the third group was any other patient visits that were lacking a correctly reported personal identification number (49,249 visits). after these exclusions, the data were aggregated, resulting in 1,534,942 eligible visits, corresponding to 1,113,341 unique patients. the first hospital visit during the study period was defined as the patient s index visit. the end of follow - up was defined as the date of death if the patient died within 1 year of the index visit, or the date 1 year after the index visit (censoring date). sex, age category, and icd-10 codes (truncated to three digits for the index visit) were obtained from the npr. to avoid the possibility of tracing back to individual patients duration in days from the index visit to end of follow - up, vital status, and history of the diseases (dichotomous variables) included in the cci that were listed up to 1 year prior to the index visit were obtained from the npr. the npr routinely obtains information on vital status and date of death from the national population register.16 the data file obtained from the npr was anonymized, and thus no requirement for regulatory ethical approval in norway was needed. the original cci included 19 diseases, but for the purposes of the present study, leukemia and lymphoma were included in the disease category any malignancy, as done by other authors.911 these 17 cci diseases were identified by the icd-10 coding defined by quan in 2005.14 a primary diagnostic code is the code for the main medical condition causing the admission. secondary diagnostic codes are codes for diseases that exist at the same time as the primary disease, or diseases that develop and need examination or treatment during the admission. the pri weights were estimated using a cox regression model of time from index visit to death with follow - up censored at 1 year, adjusted for sex, 5-year age categories (04 years an algebraically correct method was used to define new weights for the cci diseases by summing the regression coefficients, not the hazard ratios (hrs).17 the pri was constructed by multiplying the regression coefficients by a scaling constant k, then rounding it to the nearest integer, and finally summing it over all the cci diseases. the scaling constant k was chosen such that the maximum weight for a specific disease in the pri became the same integer as the maximum weight in the cci, and thus k was set to 2.3. both the construction and comparison of the pri and the cci were done through an internal tenfold cross - validation procedure.18 ten subsets of the data were randomly defined, and nine of them were used to develop the pri, with the last subset used to run the comparison. this was done for all ten subsets, and the results are given as the mean values from the ten runs. to predict 1-year mortality, three logistic regression models were fitted, one including sex and age (base model), one including sex, age, and the cci (cci model), and one model including sex, age, and the pri (pri model). model fit was compared using several measures of global fit. the bayesian information criterion (bic) is a likelihood - based measure, and for model fit, the lower the bic - value, the better the fit.19 likelihood ratio chi - square statistics were also used to test model fit. mcfadden s r is a measure of improvement in fit over the intercept model.20 the brier score was used to directly compare the observed outcomes with the predicted probabilities.19 the c - statistic, which is a summary of a model s ability to discriminate between those who do and those who do not experience the outcome, was calculated for each model.21 this measure is the most commonly used in the medical and epidemiological literature when comparing different comorbidity indices.11,13,15,22,23 the c - statistic varies from 0.5, which indicates that the discrimination is due to chance alone, to 1, which indicates perfect discrimination. the general classification of discrimination is acceptable (c [0.7, 0.8 ]), excellent (c [0.8, 0.9 ]), and outstanding (c [0.9, 1]).24 to assess which index changed the predicted value most when added to the base model, movement from the estimated probabilities of the outcome were compared. this was done using the net reclassification improvement (nri), which is defined as the difference in proportions moving up or down in risk among patients who died and those who survived. we also used the integrated discrimination improvement (idi), which measures the difference in the mean predicted probabilities between those who died and those who did not in the cci and pri models compared to the base model.25,26 we also calculated how much the probabilities changed on average when a comorbidity index was included in the model : this quantifies the effect of introducing a comorbidity index into a model. to compare the importance of including age, sex, and a comorbidity index in the model, p^ was calculated with and without each of the variables in a subanalysis with patients aged over 50 years who were not admitted to hospital due to any of the 17 cci diseases at the index visit. in the description of the disease pattern, the patient group was age - standardized according to the norwegian standard population in 2011.27 all analyses were done using stata 13.28 the majority of the 1,113,341 patients in the study were women (57.0%). for 68.1% of the patients, the index visit was a hospitalization. figure 2 presents the proportion of the norwegian population with one or more of the cci diseases in 20102011 and the total proportion admitted to norwegian hospitals in that same period. twenty - two percent of the patients were registered with at least one cci disease, but among men and women below 50 years of age, the proportion was less than 3%. for patients 50 years of age or older, the proportion with cci diseases increased with age, and the increase was more prominent among men. for both sexes, there was a strikingly high proportion of hospital admission in the youngest age - group (04 years). this was mostly due to diseases of the respiratory system (data not shown). for women, there was a peak around 3034 years (figure 2) as a result of birth - related admissions (data not shown). there was a small peak for men aged 2024 years as well, which was mostly attributed to icd-10 codes within s00-t98 (injury, poisoning, and certain other consequences of external causes, data not shown). the age - standardized proportions of the norwegian population with each of the cci diseases are shown in figure 3. the five most common cci diseases for men and women were myocardial infarction, cerebrovascular disease, chronic pulmonary disease, diabetes without chronic complications, and malignancies, but the order of importance differed. the overall proportion of deceased patients within 1 year was 4.8% (52,938 patients), and the 1-year risk of death was significantly higher for patients with at least one cci disease compared to those with none (hr 4.5, confidence interval 4.44.6) (table 1). the proportion of patients with each cci disease ranged between 0.05% (acquired immunodeficiency syndrome [aids]/human immunodeficiency virus [hiv ]) and 4.9% (chronic obstructive pulmonary disease [copd ]). metastatic tumor was associated with the highest hazard, while rheumatic diseases and diabetes without chronic complications were associated with the lowest hazard for 1-year mortality. across all cci diseases, the mean cci score was 0.4, the median number of diseases present in the patients was 0, and the range was 08. when the weights for the cci diseases in the pri were compared with those in the cci, the weights for seven diseases remained unchanged, four increased in magnitude, and six decreased in magnitude. except for two cci diseases moderate - to - severe liver disease and aids / hiv the weights in the pri only deviated by one if at all compared to those in the cci. the maximum observed cci and pri values among the patients were 12 and 15, respectively. the overall mean cci was approximately equal to the mean pri (0.43 versus 0.42). table 2 presents the measures of model fit obtained from the internal cross - validation for the three prediction models. the c - statistic was marginally better for the pri model than for the cci model (91.5 versus 91.3). all measures showed improvement in fit when the pri was included in the model instead of the cci (table 2). among patients aged 50 years or older who were not admitted to hospital due to any of the 17 cci diseases, (4.2%), and r07, pain in throat and chest (3.0%), were the two most common conditions. figure 4 shows the change in the effect of age when either of the two comorbidity indices was added to the base model for these patients. including the pri in the model led to a reduction in the effect of age for all age - groups, ranging from 3.5% in the 5559-year group to 38% in the 95 + year group. using backward elimination from the pri model for patients 50 years of age or older with an index visit that was not due to a cci disease, the change in the estimated probability (p^) was 2.8%, 4.3%, and 0.7% when the pri, age, and sex were removed, respectively. similarly, for the cci model, p^ was 2.6%, 4.7%, and 0.7% when the cci, age, and sex were removed, respectively. we observed that for patients 50 years of age or older, comorbidity based on npr data was almost equally important as age for predicting 1-year mortality. measured by the proportion of the norwegian population with a cci disease registered in the npr, the disease burden increased with age, reaching 52% and 38% in the oldest age - group for men and women, respectively. the weights for four cci diseases were higher in the pri compared to the corresponding weights in the cci, while the weights for six diseases were lower in the pri than in the cci. outstanding in discriminating, according to the standard c - statistic classification, between those who died and those who did not.24 the base model produced a c - statistic of 0.869, while the c - statistic increased to 0.913 and 0.915 when the cci and the pri were included in the model, respectively. therefore, the majority of the ability to discriminate came from sex and age, but both of the indices increased the discrimination ability further, which is in agreement with the findings of gabbe and kilgore.30 for patients over 50 years of age, we observed that the pri was almost as important as age, while the cci was relatively less important than the pri. in addition, a reduction in the effect of age for those over 50 years of age increased with age toward 38% when the pri was added to the base model, showing the importance of including information regarding comorbidity when predicting the probability of death, especially in elderly patients. the lack of data from privately financed hospitals could cause concern of possible introduced bias. however, in 2008 the total use of privately financed hospitals in norway was approximately 0.5% of the total health care service.31,32 therefore, excluding these patients would probably not have induced any major bias. the exclusion of all the outpatient visits with only medical procedures or no procedures at all probably led to an underestimation of the prevalence of the least severe diseases (those who did not need a hospitalization). the main aim in this study, however, was not to estimate the prevalence of a disease, but to construct a valid modification of the cci. if we had included all outpatient visits, this would have introduced a possible regional bias using the pri. the norwegian prescription database reported that approximately 156,000 (3.1%) of norwegians used medication for diabetes in 2011. in addition to those using medications, there is an unknown number of patients with type 2 diabetes who are managed through lifestyle changes only. however, a norwegian study reported that 4.3% of the population suffered from diabetes in the period 20062008.33 in our data, we identified approximately 47,000 cases of diabetes treated in hospitals during the study period (1% of the norwegian population). the norwegian institute of public health has reported that the prevalence of copd in norway is around 200,000 (4%).34 in our study, 54,410 patients admitted to hospital in 20102011 were either treated for copd or had it registered in their disease history. lastly, the cancer registry of norway reported a cancer prevalence of 215,000 in 2011.35 the observed prevalence from the npr is measured using a combination of prevalence and incidence, since it includes patients with either a newly diagnosed cancer or a diagnosis in the past in continuous need of treatment. however, considering any malignancy and metastatic tumor as one group in the npr data, there were 57,584 cancer patients. many patients with copd and cancer do not need an annual or biannual consultation at a hospital, either because they are well controlled or considered cured from their disease, or since their assigned general practitioner handles regular issues, explaining why the numbers in the npr from 2010 to 2011 are lower than the corresponding published prevalence estimates.33,34 bakken showed good agreement between the data included in the npr and the cancer registry of norway.36 it is reasonable to believe that the patients we identified in the npr had more severe disease and thus poorer prognosis than those suffering from the same diseases, but not admitted to hospital. the uniqueness of this study is that we used two alternative methods (nri and idi) for comparing the quality of the reclassification of patients. none of the previous papers describing modifications of a comorbidity index has to our knowledge used these predictive probability measures. the nri was 74.5% and 84.0% for the cci and pri, respectively, showing that inclusion of a comorbidity index increased the credibility of a model s predictive ability, and the model including the pri correctly determined the risk of a larger proportion of patients. also, here the model including the pri yielded a higher idi of 10.7% compared to 9.9% for the model that included the cci. therefore, the pri model has a better ability to reclassify patients than the cci model. in addition, our defined p^ illustrated the importance of including a comorbidity index in a model, as opposed to using the base model, in predicting the 1-year mortality for patients 50 years of age or older. the present study is closely related to the work done by quan and bottle and aylin.11,12 quan developed an updated version of the cci based on the population of calgary, canada, and validated it using data from six different nations. a major advantage of the study of quan was that they validated their updated index externally, in addition to being population - based. the advantage of bottle and aylin s study was that they developed new empirical weights based on english administrative data in 20072008 (with over 5 million records). however, they studied in - hospital mortality only, as individual follow - up was not possible. our proposed pri is based on much larger patient numbers than quan s, and more complete follow - up after discharge from hospital compared with that of bottle and aylin, which is very important, especially considering that only 35% of the deceased in norway in 2011 died in hospitals.37 when constructing the pri, diagnoses of the cci diseases, recorded both in primary and secondary fields from the index visit as well as the history, and recorded up to 1 year prior to the index visit were included. the rationale behind this was that such an index should be based on the most recent disease history available. pine and ghali both showed improved discrimination when conditions present upon admission were included.38,39 in all subanalyses for patients 50 years of age or older, we excluded data for the patients who were admitted to hospital due to any of the 17 cci diseases. the rationale behind this was that patients under 50 years of age have a low presence of comorbidities. in addition, we wanted to study comorbidity diseases in addition to the one causing the index visit and not morbidity. this study design is similar to the situation researchers meet when they want to add comorbidity information to their study cohort. one limitation of our study was that we were not able to validate the pri using external data. another limitation regards the inclusion criteria of patients : we excluded outpatients registered with only medical procedures or no procedures at all. this excluded many patients and limits the generalizability of the results, but it was necessary due to the large variation regarding the degree of reported personal identification numbers from private specialist practices. outpatient consultations are usually scheduled for patients with chronic and non - life - threatening diseases, such as dementia, rheumatism, and diabetes ; hence, there is a considerable risk of underestimating the number of patients suffering from these diseases in our study. on the other hand, the strengths of the study are that in contrast to the cci, the pri was developed using data from a national register. secondly, the algebraically correct method of the cci was used when developing the pri.17 finally, the large sample size and access to complete mortality data are obvious strengths. it is of high importance to include a comorbidity index in observational studies of disease prognosis, especially in elderly patients. both the cci and the pri showed a high degree of discrimination, indicating that both have good predictive ability. however, the pri explained a larger proportion of the observed effect of age, and the pri weights reflect the patterns in the data from the npr. we have shown that weighting the cci for a specific population slightly improves the performance of the cci. the pri is by its construction more representative of the general population, and can be generalized to other countries in situations where data from a national patient register are used.	objectiveto construct an updated comorbidity index (patient register index [pri ]) using national data collections from norway and compare its predictive ability of 1-year mortality with the charlson comorbidity index (cci).materials and methodsdata regarding over 1.11 million patients registered in the norwegian patient register in 2010 and 2011 were used to construct the pri. the pri was evaluated by comparing its model fit and discrimination with the cci.resultscompared with the cci, the pri weights decreased for six, increased for four, and were unchanged for seven diseases. when the pri was added to the model including age and sex, the age effects were reduced by up to 38% for patients older than 50 years. all measures of model fit improved for the pri model.conclusionadjustment for comorbidity is especially important for patients 50 years of age or older, and its effect on 1-year mortality is almost comparable to the age effect. the pri is based on more recent data than the cci, and is more representative of the general population due to its construction.
the use of dietary supplements, including multivitamins has increased substantially in the past few decades. on an average 20 - 30% of the population in developed countries use such vitamin supplements. industries involved in their manufacture are reported to be one of the world 's fastest growing industries. although, the use of multivitamin supplementation may provide benefits in terms of increased nutrient intake, there are potential adverse effects also due to high intake. although certain benefits, like that of folic acid supplementation and protection against neural tube defects in specific populations is well - established, but results of some large scale randomized trials have shown that for the majority of the population multivitamin supplements are ineffective. sesso. in a randomized controlled trial conducted in 14,641 participants in usa revealed that daily multivitamin supplement did not reduce cardiovascular events, myocardial infarction or stroke in men. in today 's scenario with more literate and health conscious patients who are capable of making their own decisions regarding their health care coupled with wide availability of such supplements, present study was conducted to explore pattern of use, public knowledge and attitude toward consumption of these multivitamin supplements. a descriptive cross - sectional population based study was conducted from january to june 2013 among the general public. subjects included were patients and other hospital attendees like patients ' family members / relatives or friends, of either sex aged 18 years or older while they were waiting in the outpatient department. study participants were assured of confidentiality and anonymity of the information and written informed consent was obtained from them. a total of 120 participants were interviewed and information was collected in a structured questionnaire. the questionnaire was based on previous studies undertaken among adults about their attitude toward multivitamin supplementation and it was suitably modified for the present setting. the survey questions in addition to questions covering demographic, education level (primary, secondary or above), alcohol drinking status, contained questions regarding awareness of multivitamin supplements, its consumption (frequency, duration of usage), reasons for usage (self - medication, physician advice) and its effect. at the end of the study pearson chi - square test was applied to see the association between genders and educational status with the use of multivitamins. of the 120 study participants, 66 were males (55%) and 54 were females (45%). mean age of the males was 43.85 15.44 years and of females was 38.75 12.87 years. no association was found between gender and use of multivitamins (p = 0.969). regarding educational status, approximately (58) 48% were graduates, (13) 11% postgraduates, (36) 30% had completed secondary education and (7) 6% had covered only primary education while (6) 5% were illiterate. educational status of the participants did not show any association with the use of multivitamins (p = 0.583). source of awareness of vitamin supplement use was a family doctor, relatives or friends and media, e.g. newspaper / internet [table 1 ]. percentage of participants with different sources of information regarding multivitamins about 68.33% (82) of the participants were either current or former users of multivitamin supplements while 31.66% (38) claimed that they had never taken multivitamins. out of these users, 18.2% (15) admitted taking multivitamins of their own, 69.5% (57) consumed on the advice of their physicians while 12.1% (10) relied on the advice of family or friends. among the users, 70.73% (58) considered multivitamin supplements to be helpful. study respondents when queried as to whether they had experienced any health problem due to the use of such supplements, only one reported diarrhea which he believed to be related to multivitamin supplement use. majority of the participants were unaware of the harmful effects of multivitamin supplements or their possible interactions with other drugs. nearly 12% (14) of study participants were alcoholics and all of them were multivitamin supplement users. out of these, only three said that there is a benefit of taking these supplements along with alcohol while seven considered no benefit in consuming supplements along with alcohol. reasons quoted for the self - medication use of multivitamins are depicted in figure 1. physician prescribed reasons were multiple, like in mouth ulcers, with concurrent antibiotic prescription, arthritis, hypotension, weakness, leg pain etc. reasons for the use of multivitamin supplements among self - medicated users there was a variable response regarding frequency of vitamin supplementation. out of the self - medication group, approximately 10 consumed multivitamin on a daily basis while 3 consumed 2 times a day. some of them reported consuming them 4 times weekly or some even mentioned taking for 4 weeks after every 6 months [table 2 ]. frequency of usage of multivitamins by self - prescribed users majority of the participants were unaware regarding the correct indications for the multivitamin supplementation. study participants who were aware of the multivitamin use when asked about the opinion whether these drugs can be self - medicated, 60% (50) responded affirmatively. regarding knowledge about the natural sources of these vitamins, as many as 76% (64) showed ignorance. in the present scenario of high prevalence of nutrient supplementation use world - wide, present study was conducted to examine multivitamin consumption in the general adult population. our study depicted multivitamin use by 68.33% of the participants. similarly, national health and nutrition examination survey 2003 - 2006 reported use of dietary supplements by 53% of the respondents and out of which most frequently used supplement was multivitamin. a survey conducted in columbia on the estimate of use of dietary supplements revealed 73% users, out of which 85% reported using multivitamin supplements. likewise, a study conducted in 11,929 men in germany reported use of vitamin or mineral supplements by 40% of population. moreover, internet sites and television are flooded with advertisements depicting increased energy with the use of such supplements. health conscious people now - a - days feel happy to consume such supplements thinking they are actively taking care of their health. most common source of information regarding vitamin supplement use, in our study was doctors, 69.1% users consumed multivitamins on the advice of doctors. these results are in concordance with the study conducted in karachi, which revealed recommendation of vitamin supplement by doctors to be 66.2%. number of self - prescribed multivitamin users consuming multivitamins on a daily basis was high which is consistent with results from other studies where dietary supplement was consumed on a daily basis by more than 50% of multiple dietary supplement users. this is an area of concern as it has been seen in some other studies as well. a study conducted in america among five ethnic groups found that the median daily nutrient intake from multivitamin / minerals among users were above the recommended daily allowance for some vitamins such as a, b-6, b-12 and e, thiamin, riboflavin, niacin, pantothenic acid and folate. this is more so a problem in the country like ours where there is the wide availability of supplements without physician prescription, which can clearly lead to excessive use of vitamins and hence possible adverse effects or interactions. furthermore, nutrient composition of multivitamins is extremely variable which in some cases can lead to excessive use. in india, chughchugh and lhamo have shown that the majority of the supplements available in the market contained nutrient amounts higher than the recommended dietary intakes. various studies such as alpha - tocopherol, beta - carotene cancer prevention study reported increased risk of hemorrhagic stroke by 50% with the use of alpha tocopherol for 6 years and selenium and vitamin e cancer prevention trial revealed that vitamin e supplements could increase the risk of prostate cancer among healthy men. reasons quoted for using vitamin supplements by self - prescribers were similar to study conducted by dickinson. where maintaining wellness was the top reason for personal use. similar reasons were seen in studies by neuhouser and eldridge and sheenan though there is no conclusive evidence that vitamin supplementation is beneficial in persons with adequate dietary intake. results of the meta - analysis have revealed that for the majority of the population multivitamin supplements are not effective. notable ones are meta - analysis of five randomized control trials which found no significant beneficial effect of such supplementation and data from eight prospective studies did not support the hypothesis that use of folate, vitamin a, c, e and multivitamins reduce the risk of lung cancer. ignorance of the participants regarding potential deleterious effects of over supplementation reflected their false belief that these medications are safe. hence, physicians should ask direct questions to their patients regarding use of self - prescribed multivitamins and educate them toward harmful effects and potential interactions with prescription medication. interaction between vitamin e and aspirin leading to additive antithrombotic effect and between vitamin e and warfarin leading to increased risk of bleeding are available in the literature. similarly, large doses of antioxidants such as vitamin e and alpha lipoic acid can decrease the effectiveness of radiation or chemotherapy for cancer. there are certain limitation of this study, which needs to be acknowledged like its cross - sectional design and its conductance at the single hospital. furthermore, association between different variables can not be established as it is a descriptive cross - sectional study. findings from this study suggest that multivitamin use is highly prevalent and the majority of the participants were ignorant of any possible harm or drug interactions. in light of this, there is a need to adopt certain educational interventions to minimize self - directed supplement use. at the same time health care professionals should take extra care to know about their patients multivitamin use and hence should counsel them about the correct use.	background and objective : the use of supplements has increased substantially in the past few decades. the present study is an effort to explore pattern of use, knowledge and attitude toward consumption of multivitamin supplements among the general public.materials and methods : a descriptive cross - sectional study on 120 adult participants from the general public was conducted. the participants were interviewed and information was collected in a predesigned structured questionnaire. the data was analyzed and expressed as counts and percentages.results:of the 120 study participants, 66 were males and 54 were females. results revealed that 68.33% (82) of the participants were users of multivitamin supplements. out of the users, 69.5% (57) participants consumed on the advice of doctors, 18.2% (15) were self - prescribers while 12.1% (10) relied on advice of family or friends. among the users, 70.96% considered such supplements to be helpful. reasons quoted for self - medication use of multivitamins were multiple such as maintenance of general health (55%), to allay weakness or fatigue (20%), to improve appetite (15%) etc. majority of the participants were unaware regarding the correct indications for multivitamin supplementation. regarding knowledge about the natural sources of these vitamins, as many as 76% showed ignorance.conclusion:finding from this study suggests that multivitamin use is highly prevalent and the majority of the participants were ignorant of any possible harm or drug interactions. in light of this, there is a need to adopt certain educational interventions to minimize self - directed supplement use and increase awareness regarding their correct usage.
approval from our institutional review board was obtained for this retrospective study. at our institution, the patients with a history of acl reconstruction surgery undergo mri when they have persistent, recurrent or new symptoms or they have re - injured their knee. after clinical examination and reviewing the mr images, follow - up arthroscopic examinations were performed for patients suspected of having a torn acl graft or other internal derangement of the knee. a computer search of the mri examinations that were done at our hospital from june 2000 to march 2007 yielded 120 consecutive patients who underwent mri of the knee after acl reconstruction surgery. finally, 51 consecutive mr examinations in 48 patients (40 men and 8 women, age range : 18 - 60 years, mean age : 32 years) were included in this study. the mr examinations were performed at a mean of 31 months (range : 4 - 192 months) after the initial acl reconstruction surgery. the mean time interval between the postoperative mr examinations and the follow - up arthroscopy was 50 days (range : 0 day-9 months). the arthroscopic records revealed 26 cases with intact acl grafts, 12 with partially torn acl grafts and 13 with completely torn acl grafts. the graft materials we used were autogenous quadriceps tendon (n = 39), autogenous bone - patellar tendon - bone (n = 9) and allogenous achilles tendon (n = 3). mr examinations were performed on 1.0-t or 1.5-t mr scanners (siemens, erlangen, germany). the mri protocols included the sagittal spin echo t1-, the turbo spin echo t2- and the proton density - weighted images, the coronal turbo spin echo t2- and the proton density weighted images and the oblique coronal turbo spin echo t2-weighted images. the oblique coronal t2-weighted images were obtained in the plane parallel to the course of the femoral intercondylar roof on the sagittal scout images (fig. the parameters of the routine knee mri were as follows : tr / te = 500/12 (t1-weighted image), 3500/15 or 2200/14 (proton density weighted image), 3500/98 or 2200/90 (t2-weighted image), a 4-mm slice thickness, a 0.2-mm interval and a 256256 or 512512 matrix. the oblique coronal t2-weighted image parameters were as follows : tr / te = 3000 - 4000/96, a 3-mm slice thickness, a 0.15-mm interval, a 256256 or 512512 matrix and a 3-minute 8-second or 3-minute 55-second acquisition time. the images were retrospectively reviewed by two musculoskeletal radiologists with 14 years and nine years of experience, respectively. the imaging sequences were grouped into two evaluation sets by a radiologist who was not involved in imaging interpretation. initially, each reader independently evaluated the status of each acl graft with using the routine knee mr images only (imaging group a) and then with using the routine knee mr images in combination with the oblique coronal images (imaging group b). the severity of acl graft injury was graded using a 3-point system ; i.e., grades 0, 1, 2 (1, 4). grade 0 implied an intact graft, grade 1 a partial tear and grade 2 a complete tear. we regarded an intact graft as a low signal intensity graft with or without longitudinally increased signal intensity streaks, well - preserved continuation and a taut orientation. some grafts with focal or rarely diffuse increased signal intensity or a slight lax orientation were included as intact grafts (2). on the other hand, hyperintensities were almost equal to fluid or graft thinning in the acl grafts on the t2-weighted images were considered suggestive of a partial or full thickness graft tear (13, 14). to differentiate grade 1 and 2 injuries, a near full - thickness defect, the lack of continuity or an indistinct ligament contour were considered indicators of grade 2 injury (fig. in addition to assessing the severity of graft injury, the two readers were requested to assign a confidence level for the diagnosis in the two imaging groups based on a 5-point scale, that is, 1 : completely uncertain, 2 : small likelihood, 3 : equivocal, 4 : probable and 5 : very certain. weighted kappa statistics were used to assess the diagnostic agreement between the mri diagnoses and the arthroscopic results of the two imaging groups (11). the strength of agreement was interpreted according to the guidelines described by landis and koch (15), that is, 0 : poor, 0.01 - 0.20 : slight, 0.21 - 0.40 : fair, 0.41 - 0.60 : moderate, 0.61 - 0.80 : substantial and 0.81 - 1.00 : almost perfect. confidence levels for interpretation were scored for each imaging group, and the difference in the confidence levels between the two imaging groups was assessed using the paired t - test. the sensitivity, specificity and accuracy for detecting graft tear were calculated by grouping the grade 1 and 2 injuries. approval from our institutional review board was obtained for this retrospective study. at our institution, the patients with a history of acl reconstruction surgery undergo mri when they have persistent, recurrent or new symptoms or they have re - injured their knee. after clinical examination and reviewing the mr images, follow - up arthroscopic examinations were performed for patients suspected of having a torn acl graft or other internal derangement of the knee. a computer search of the mri examinations that were done at our hospital from june 2000 to march 2007 yielded 120 consecutive patients who underwent mri of the knee after acl reconstruction surgery. finally, 51 consecutive mr examinations in 48 patients (40 men and 8 women, age range : 18 - 60 years, mean age : 32 years) were included in this study. the mr examinations were performed at a mean of 31 months (range : 4 - 192 months) after the initial acl reconstruction surgery. the mean time interval between the postoperative mr examinations and the follow - up arthroscopy was 50 days (range : 0 day-9 months). the arthroscopic records revealed 26 cases with intact acl grafts, 12 with partially torn acl grafts and 13 with completely torn acl grafts. the graft materials we used were autogenous quadriceps tendon (n = 39), autogenous bone - patellar tendon - bone (n = 9) and allogenous achilles tendon (n = 3). mr examinations were performed on 1.0-t or 1.5-t mr scanners (siemens, erlangen, germany). the mri protocols included the sagittal spin echo t1-, the turbo spin echo t2- and the proton density - weighted images, the coronal turbo spin echo t2- and the proton density weighted images and the oblique coronal turbo spin echo t2-weighted images. the oblique coronal t2-weighted images were obtained in the plane parallel to the course of the femoral intercondylar roof on the sagittal scout images (fig. the parameters of the routine knee mri were as follows : tr / te = 500/12 (t1-weighted image), 3500/15 or 2200/14 (proton density weighted image), 3500/98 or 2200/90 (t2-weighted image), a 4-mm slice thickness, a 0.2-mm interval and a 256256 or 512512 matrix. the oblique coronal t2-weighted image parameters were as follows : tr / te = 3000 - 4000/96, a 3-mm slice thickness, a 0.15-mm interval, a 256256 or 512512 matrix and a 3-minute 8-second or 3-minute 55-second acquisition time. the images were retrospectively reviewed by two musculoskeletal radiologists with 14 years and nine years of experience, respectively. the imaging sequences were grouped into two evaluation sets by a radiologist who was not involved in imaging interpretation. initially, each reader independently evaluated the status of each acl graft with using the routine knee mr images only (imaging group a) and then with using the routine knee mr images in combination with the oblique coronal images (imaging group b). the severity of acl graft injury was graded using a 3-point system ; i.e., grades 0, 1, 2 (1, 4). grade 0 implied an intact graft, grade 1 a partial tear and grade 2 a complete tear. we regarded an intact graft as a low signal intensity graft with or without longitudinally increased signal intensity streaks, well - preserved continuation and a taut orientation. some grafts with focal or rarely diffuse increased signal intensity or a slight lax orientation were included as intact grafts (2). on the other hand, hyperintensities were almost equal to fluid or graft thinning in the acl grafts on the t2-weighted images were considered suggestive of a partial or full thickness graft tear (13, 14). to differentiate grade 1 and 2 injuries, a near full - thickness defect, the lack of continuity or an indistinct ligament contour were considered indicators of grade 2 injury (fig. in addition to assessing the severity of graft injury, the two readers were requested to assign a confidence level for the diagnosis in the two imaging groups based on a 5-point scale, that is, 1 : completely uncertain, 2 : small likelihood, 3 : equivocal, 4 : probable and 5 : very certain. weighted kappa statistics were used to assess the diagnostic agreement between the mri diagnoses and the arthroscopic results of the two imaging groups (11). the strength of agreement was interpreted according to the guidelines described by landis and koch (15), that is, 0 : poor, 0.01 - 0.20 : slight, 0.21 - 0.40 : fair, 0.41 - 0.60 : moderate, 0.61 - 0.80 : substantial and 0.81 - 1.00 : almost perfect. confidence levels for interpretation were scored for each imaging group, and the difference in the confidence levels between the two imaging groups was assessed using the paired t - test. the sensitivity, specificity and accuracy for detecting graft tear were calculated by grouping the grade 1 and 2 injuries. the mr grades of the acl graft injury for each reader and each imaging group are summarized in tables 1 and 2, respectively. the diagnostic agreements between the mr grade and the arthroscopic grade for imaging group a were considered " moderate " with weighted kappa values of 0.555 (reader 1) and 0.515 (reader 2). on the other hand, those for imaging group b were " substantial " with weighted kappa values of 0.666 (reader 1) and 0.611 (reader 2). between the two imaging groups, a mismatch was noted for seven cases (downgrading for 6 and upgrading for 1) by reader 1 ; there were mismatches for eight cases (downgrading for 5 and upgrading for 3) by reader 2. the readers reached the correct diagnoses in eight cases (5 cases by reader 1 and 3 cases by reader 2) of the 11 cases for which they downgraded an acl graft injury on the oblique coronal imaging, and the arthroscopic results were seven cases with an intact graft and one case with a partially torn graft (figs. 3, 4). in three cases (1 case by reader 1 and 2 cases by reader 2) of the four upgraded cases, the correct diagnoses were achieved on the oblique coronal imaging and the arthroscopic results were two cases with a partially torn graft and one case with a completely torn graft. interobserver agreement between the two readers was considered " substantial " with weighted kappa values of 0.614 (imaging group a) and 0.730 (imaging group b). the confidence level for imaging group b was significantly higher than that for imaging group a (p < 0.01). the mean confidence level for the correct diagnosis was higher than that of the incorrect diagnosis for each reader and each imaging group (p < 0.01). the overall mr sensitivity, specificity and accuracy for the diagnosis of acl graft tear were calculated by combining grade 1 and 2 injuries as tear (table 4). the imaging of group b had higher specificity and accuracy than did the imaging of group a for each reader. in our study, the diagnostic accuracy for acl graft injury was improved by the addition of oblique coronal imaging to the routine knee mr sequences. the overall mr specificity and diagnostic accuracy for acl graft tear oblique coronal imaging may lead mr readers either to downgrade or upgrade the acl graft injury that is originally seen on routine knee mri, and so this helps reach a correct diagnosis. the mr readers were more confident of graft assessment with viewing the additional oblique coronal imaging than by viewing the routine knee mri alone. (16) recommended using the oblique coronal mri for visualizing the anatomic diagonal course of the native acl and its relation with the intercondylar notch and the posterior cruciate ligament. for the native acl, the use of additional oblique coronal images improves the specificity and accuracy for detecting acl tear and this also raises the accuracy of grading acl injury (11). for the acl graft, one previous study included the oblique coronal images for the evaluation of healthy acl grafts (7). the reasons why the diagnostic accuracy was enhanced by the additional oblique coronal images in our study are presumed to be as follows. first, the full length of an acl graft can be viewed in a single plane along its anatomic diagonal course, and thus, the graft is less subject to volume averaging. second, the transverse width of an acl graft can be easily appreciated on the oblique coronal images because both the medial and lateral margins of the graft are clearly visualized. third, the oblique coronal imaging reduces paramagnetic artifacts by avoiding fixation devices in the plane, while the artifacts from metallic fixation screws obscured the femoral and tibial bone tunnels on orthogonal sagittal and coronal images. the majority of the previous mr studies have employed sagittal, coronal or oblique sagittal images (1 - 6, 17). some investigators have conducted mr studies using proper knee positioning in order to optimize visualization of an acl graft (18). contrast - enhanced mr studies have been performed to evaluate the periligamentous tissue with its higher signal intensity, and this higher signal intensity was derived from neovascularization, granulation tissue or immature collagen (19 - 21). two previous studies evaluated the oblique axial images obtained at a right angle to the acl graft (8, 9), and one report used mr arthrography for acl graft assessment (22). these studies have shown various accuracies for conventional mri ; the largest of these studies on 52 patients, and two of these 52 patients had torn grafts, showed 100% sensitivity, 86% specificity and 86.5% accuracy for detecting a tear (17). the diagnosis of partial tear of an acl graft is more difficult than that of complete tear : on a conventional mr study of 16 patents, the diagnosis of partial tear versus other conditions (intact graft or complete tear) resulted in 0% sensitivity, 67% specificity and 37.5% accuracy (1). similar in our study, false positive or negative diagnoses of partial tear were the main causes of the relatively low accuracy for the overall grading of acl graft injury on mri. the readers found it difficult to correctly ascertain a normal or partially torn graft when a subtle abnormality was suspected within the graft on mr images. using the oblique coronal images reduced the false - positive diagnosis of partial tear and increased the specificity of mri for acl graft injury. nevertheless, false negative diagnoses for acl graft injury were still made with using the oblique coronal images. we believe that the femoral attachment site is vulnerable to misinterpretation because of the acute angle formed between the femoral tunnel and the grafts on the oblique coronal images. the oblique sagittal images may help improve the visualization of the femoral attachment site of an acl graft by showing the femoral tunnel in a plane (2). in other cases, a slightly attenuated, lax acl graft at arthroscopy had a normal appearance on mr images. the readers made many false negative diagnoses even with using the oblique coronal images because the mr diagnoses were based on the morphologic abnormalities, and there was insufficient information on such functional abnormalities as graft laxity. for these cases, we may reduce the rate of a false negative mr diagnosis by referring to the clinical data. some limitations of the current study should be mentioned. first, our study population included only a small number of patients with partial or complete acl graft tears. second, the time interval between the mr examination and arthroscopy, as well as the chronicity of graft tear, which might have affected the mr findings, were disregarded in our study. third, because the weighted kappa statistic does not provide a p value, we could not calculate the statistical significance of the difference between the diagnostic accuracies of the two imaging groups. in conclusion, our results suggest that the use of additional oblique coronal images improves both the diagnostic accuracy and the diagnostic confidence for grading acl graft injury. using the oblique coronal images also increases the specificity and accuracy of acl graft tear, and this may help physicians make correct clinical / surgical decisions when treating these ligament injuries.	objectivethe purpose of this study was to evaluate the diagnostic efficacy of using additional oblique coronal mri of the knee for grading anterior cruciate ligament (acl) graft injury after ligament reconstruction surgery.materials and methodswe retrospectively reviewed 51 consecutive mr knee examinations of 48 patients who underwent both acl reconstruction and follow - up arthroscopy. the mr examinations included the orthogonal axial, sagittal, coronal images and the oblique coronal t2-weighted images, which were oriented in parallel with the course of the femoral intercondylar roof. two radiologists independently evaluated the status of the acl grafts with using the routine knee mri and then with adding the oblique coronal imaging. the severity of acl graft injury was graded using a 3-point system from mr images as intact, partial tear or complete tear, and the results were compared with the arthroscopic results. weighted kappa statistics were used to analyze the diagnostic accuracies of the knee mri with and without the additional oblique coronal imaging. for each evaluation, the observers reported a confidence level for grading the acl graft injuries in the two imaging groups.resultsthe weighted kappa values according to the routine knee mri were 0.555 (reader 1) and 0.515 (reader 2). the inclusion of additional oblique coronal imaging increased the weighted kappa values to 0.666 (reader 1) and 0.611 (reader 2). the mean confidence levels by each reader were significantly higher (p < 0.01, paired t - test) with the additional oblique coronal imaging than by using the routine knee mri alone.conclusionthe additional use of oblique coronal mri of the knee improves both the diagnostic accuracy and confidence for grading acl graft injury.
head and neck cancer is the sixth most common cancer and is responsible for almost 200,000 deaths around the world each year [13 ]. in the united states, head and neck squamous cell carcinoma (hnscc) accounts for more deaths annually than cervical cancer, melanoma, or lymphoma. although recent molecular studies have advanced our understanding of the disease and provided a rationale for the development of novel therapeutic strategies, hnscc is still associated with severe mortality. in addition, the 5-year survival rate is even lower for hnscc patients with a single homolateral lymph node metastasis (lnm) and is less than 25% for patients with bilateral lnm. understanding the biology of hnscc, progression will greatly assist in treatment decisions and in the development of new strategies for prevention and control of this disease. currently, the progression of hnscc is considered to result from evolution through stepwise alterations in multiple molecular and cellular pathways [8, 9 ]. however, this evolution concept has limitations in explaining the heterogeneity observed in a single tumor nest. it has been known for a long time that there are subpopulations of cells within solid tumors that contain different biological behaviors, such as metastatic potential [10, 11 ]. accumulating evidence supports the subpopulation observation, particularly, the existence of so - called cancer stem cells (cscs) [1217 ]. although cscs in solid tumors including hnscc have not been precisely identified, the csc hypothesis opens a new era in understanding the initiation and progression of cancers. this short review will briefly introduce the csc concept, summarize the current progress of csc studies in hnscc, and discuss the potential application of the csc concept to the clinical management of hnscc. cscs are defined as a small subset of cancer cells that constitute a pool of self - sustaining cells with the exclusive ability to maintain the tumor. currently, there are two hypothetical explanations for the existence of cscs. cscs may arise from normal stem cells by mutation of genes that render the stem cells cancerous. or, they may come from differentiated tumor cells that experience further genetic alterations and, therefore, become dedifferentiated and acquire csc - like features. if the csc hypothesis is true, many aggressive behaviors of cancer cells, such as chemoresistance and metastasis, may be better understood. current csc research is focusing on the identification of csc in solid tumors, since stem cells in hematopoietic malignancies such as leukemia have been well characterized [1216 ]. however, many difficulties are encountered when exploring the existence of cscs in solid tumors, due to the inaccessibility of tumor cells and the lack of appropriate functional assays. an important breakthrough in the study of solid tumor cscs was the identification of breast cancer cscs and their biomarkers by clarke and his colleagues in 2003. since then, cscs have been reported in neoplasms of brain, prostate, lung, colon, pancreas, liver, melanoma, and skin [1933 ]. among them, the breast csc model with well - defined biomarkers is more advanced than in other types of cancers [3436 ]. using this model, molecular signatures and signaling there are three main characteristics that define cscs : (1) differentiation, which provides the ability to give rise to a heterogeneous progeny, (2) self - renewal capability that maintains an intact stem cell pool for expansion, and (3) homeostatic control that ensures an appropriate regulation between differentiation and self renewal according to the environmental stimuli and genetic constraints of each organ tissue, which accounts for the tissue specificity of cscs. currently, xenograft assays for different organ sites have been established for testing cscs. as suggested by the aacr workshop on cancer stem cells in 2006, this type of assay allows reliable testing for all three characteristics of cscs. in current studies, cancer cells from either tumor tissues or cell lines are initially sorted by specific cell surface markers. the selected cell population is then injected into experimental animals for tumorigenesis testing. if as few as 100500 cells of the selected cell population are tumorigenic, the featured cell surface markers can serve as csc - specific biomarkers. in a breast cancer study by al - hajj., human breast cancer tissues or cells with or without expression of cd44 and cd24 were injected into the mammary fat pad of immune - deficient nonobese diabetic / severe combined immune - deficient (nod / scid) mice, which have greater immune deficiency than nude mice. using this model, similar xenograft assays in nod / scid mice were used to identify cscs of brain, colon, and lung with a cd133 profile [19, 21, 3941 ]. not only the nod / scid mouse models but also nude mice are choices for an orthotopic xenograft assay. visvader and lindeman have recently summarized mouse models and csc markers used for isolation of csc, including cd133, cd44, aldh1a1, and epithelial cell adhesion molecule (epcam). as shown in table 1, there is no universal csc marker for all types of cancer. csc markers may be tumor type specific, depending on the niche of each type of csc. in addition to in vivo assays for csc identification, many in vitro experiments have also provided evidence for the existence of cscs. for example, studies by collins. focused on a cell population in patients ' tumor tissues featuring cd44/integrin21/cd133. these cells were examined by colony - formation and long - term serial culture assays and showed self renewal and regeneration of phenotypically mixed populations. accumulating evidence suggests that cscs contribute not only to tumor initiation, but also to aggressive tumor behaviors such as chemoresistance and metastasis. it has been noted that although chemotherapy kills the majority of cancer cells in tumor tissues, it may leave a population of cells behind. these cells overexpress the atp - binding casstte (abc) drug transporters which protect cancer cells from damage by cytotoxic agents. coincidently, a side population (sp) of tumor cells which are defined by their inability to accumulate the fluorescent dye hoechst 33342 due to overexpression of the abc transporter abcg2 has been confirmed to hold csc features in several types of cancers including hematopoietic, prostate, and glioma cscs [4244 ]. abcg2 and other abc transporter proteins, therefore, have served as csc markers (table 1). for example, a study of a colorectal cancer cell line that is resistant to 5-fluorouracil (5fu) and oxaliplatin by dallas. showed 5- to 22-fold enrichment of a double csc marker cd133/cd44 population. another study by hermann. showed that human pancreatic cells that survived prolonged treatment with gemcitabine had a 50-fold increase in a cd133 population. considering cscs a target population for the treatment of human cancer has opened new directions for research efforts in the field. the development of inhibitors against the abc transporter abcg2 has been explored in clinical studies. on the other hand, targeting specifically activated signaling pathways in cscs may provide an effective strategy to eliminate this cell population. dallas. reported that chemoresistant colorectal cancer csc - like cells showed increased expression of insulin - like growth factor-1 receptor (igf-1r). this cell population responded to inhibition by an igf-1r monoclonal antibody more effectively than its nonresistant counterpart. several signaling pathways, including the wnt, tgf-, and cxcr4 pathways, have been suggested to be activated in cscs [17, 48, 49 ]. therapeutically targeting these pathways deserves further investigation. the existence of mcscs was first hypothesized in 2005 by brabletz., based on their observations in colorectal cancer they proposed that scscs are embedded in epithelial tissues or epithelial - based tumors and can not disseminate. in contrast, mcscs, which are derived from scsc by acquiring a transient epithelial - mesenchymal transition (emt), are located at the tumor - host interface and mediate tumor cell metastasis. in a colorectal cancer model, brabletz. observed that not only the expression levels of emt - related biomarkers but also their locations in the tumor nest were significantly associated with metastasis. they found that loss of e - cadherin (e - cad) usually resulted in nuclear localization of -catenin, which is a typical feature of emt, and nuclear -catenin was accumulated in dedifferentiated tumor cells at the tumor - host interface. the authors then interpreted these observations in the context of the scsc and mcsc hypotheses, suggesting that scsc and mcsc are responsible for formation of the primary tumor and metastasis, respectively. both scsc and mcsc particularly, metastatic tumors generated from mcsc may experience a mesenchymal - epithelial transition (met) in the metastatic organ site, which may explain why emt can not be clearly observed pathologically in many metastatic lesions. in fact, the mcsc hypotheses can be used to explain the heterogeneous morphology of the primary tumor and how metastases can recapitulate the heterogeneity in differentiation and tumor - cell dormancy and disease recurrence. mani. reported that the stem - like cells identified in breast cancer were associated with emt markers [49, 52 ]. a cd133/cxcr4 stem - like population isolated by hermann. was suggested to be essential for metastasis of pancreatic cancer [32, 53 ]. although the concept of developmental hierarchy of solid tumors has been discussed in several papers, the hypothetical hierarchical model of csc / progenitors was clearly proposed in 2007 by tang. based on their studies in prostate cscs [43, 54 ]. this model described a hierarchical organization of phenotypically and functionally distinct cells at different stages of prostate tumor maturation. their study demonstrated that a highly purified cd44 population was still heterogeneous and enriched in tumorigenic and metastatic progenitors. that is, not only csc but also progenitors can be tumorigenic in the nod / scid mouse model. these two types of tumor cells share the common marker cd44, but they can be distinguished by other well - defined markers including abcg2 and 21, which are specific for tumor progenitors. recently, identified chromosomal instability that usually supports a stochastic model in the mcsc population isolated from liver metastasis of colon cancer. they, therefore, proposed a new model which suggested that both stochastic and hierarchical models can be used to explain the mcsc population (figure 1). to date, only a few studies of hnscc csc have been reported. using both nod / scid mice and rag2/cytokine receptor common -chain double knockout (rag2dko) mice, prince., the same group that identified breast cscs, reported that as few as 5 10 cd44 hnscc cells could generate tumors in the mice and demonstrated tumor heterogeneity. examining samples from human hnscc tissues revealed that the cd44 population varied from 0.1% to 41.7%. this cell population also inclusively expressed bmi1, a nuclear protein that also plays a role in self renewal in other cscs, while exclusively expressed the differentiation marker involucrin. unlike breast cscs, this group found that epithelial - specific antigen (esa) expression was not enriched in the tumorigenic cells, suggesting that hnscc has csc biomarkers distinct from those in breast cancer. a cd44 population was also reported by okamoto. to characterize hnscc csc - like cells. it was found that cd44 cells possessed not only a capacity for forming tumor spheres, proliferation, migration, and invasion in vitro, but also a resistance to chemotherapeutic agents. supporting this observation, four relevant chemoresistant genes, abcb1, abcg2, cyp2c8, and tert,, an sp was identified by zhang., and proved to enhance the capability of tumor formation in nude mice as compared with non - sp. in another study, oral cancer stem - like cells were enriched through sphere formation and found to express oct-4, nanog, cd133, and abcg2. nanog / oct-4/cd133 triple - positive status predicted a poor prognosis for patients with oral cancer. cd133 is also reported as an hnscc stem - like cell marker by studies using a head and neck cancer cell line. these data can be supported by many observations showing that a small population of hnscc tumor cells exists and demonstrates strong self - renewal and proliferation capabilities, even in the early stage of tumor development [6264 ]. in tumor cells of epithelial origin, in fact, this tumor subpopulation is also responsible for more aggressive phenotypes, such as resistance to cancer therapeutic drugs and metastasis [50, 51 ]. whether putative cscs play a role in metastasis of hnscc or not the existence of mcsc has not been reported. we found that a highly metastatic subpopulation selected from a xenograft mouse model expressed high levels of csc markers, including cxcr4 and integrin 1, and altered levels of emt markers such as e - cadherin and vimentin [6567 ]. cxcr4 has been investigated as a putative csc marker and is also an ideal target for the treatment of metastatic hnscc. integrin 1 is mainly expressed in the basal layer of the normal epithelium as an epithelial stem cell marker [64, 68 ]. in abnormal epithelium (hyperplasia and dysplasia), integrin 1 is found to be expressed in the upper layers of the epithelial tissues. it is also expressed in a variety of tumor tissues. integrin 1 overexpression has been suggested to expand the csc compartment by inhibiting differentiation and apoptosis, therefore contributing to tumor progression and metastasis. a recent study by kirkland and ying showed that 21 integrin regulated lineage commitment in multipotent human colorectal cancer cells. whether the metastatic populations contain csc - like features or not is currently under investigation. from a clinical perspective, if the csc or csc - like population represents the more aggressive hnscc population, the early detection and targeted treatment of these cells become an urgent need in order to better manage this disease. an immunohistochemistry study of primary hnscc reported by prince and ailles showed that cd44 staining was associated with more basal - appearing cells. cd44 cells were costained with markers for the basal normal squamous epithelium, ck5/14, while cd44 cells were associated with the differentiation marker involucrin, supporting the organization of hnscc by developmental hierarchy, as predicted by the csc theory of carcinogenesis. however, some studies of cd44 as a csc marker in human hnscc tissues contradict these in vitro and in vivo studies. a recent study by mack and gires reported cd44s and cd44v6 expressions in head and neck epithelial tissues. they found a similarly high level of cd44s and cd44v6 expression in normal, benign, and malignant epithelia of the head and neck. therefore, the value of cd44s as a marker for a small csc population in hnscc needs to be reconsidered. we believe that there is a necessity to precisely define more hnscc csc markers with an aim of further improving our ability to isolate hnscc cscs. aldh1 has been considered a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. expression of aldh1 in hnscc and dysplastic mucosa tissue samples was examined by visus.. they found that 12 of 17 hnscc and 30 of 40 dysplastic mucosa tissues expressed this protein. however, this study did not correlate aldh1 expression status with aggressiveness or prognostic features of the disease, such as metastasis, chemoresistance, or survival. our recent study of hnscc tissues demonstrated a statistically significant increase in aldh1 expression in tumors with lnm compared to tumors without lnm (p <.0003, figure 2). although aldh1 has not been reported as a marker for hnscc csc, our study suggests that aldh1 may be a potential marker for tumor progression and metastasis in hnscc. in addition to their predictive and prognostic value, the identification of cscs in hnscc will also provide target populations that require more aggressive treatment than can be achieved with conventional therapies, such as a combination treatment with chemotherapy and an agent targeting csc - specific signaling pathways. as discussed in section 3.1., a combination of chemotherapy with inhibitors of the abc transporters overexpressed by cscs may have potential clinical application. furthermore, recent progress in nanotherapeutics has shown the ability of nanoparticles to bypass abc transporters when delivering anticancer drugs to tumor cells, providing a new strategy to overcome chemoresistance of cscs. recent progress in the study of cscs in solid tumors has provided researchers and clinicians in head and neck cancer new concepts to better understand the heterogeneity of this disease with. once csc or csc - like populations are defined with appropriate biomarkers, these biomarkers can be used for accurately detecting tumor - initiating cells or metastatic cells in primary tumor biopsies, which will aid clinicians in their treatment decisions and in the accurate prognosis of hnscc. currently, there are no consistently well - defined biomarkers or matured technologies to identify csc or csc - like populations in hnscc. efforts are being made to improve this situation by developing in vitro models and appropriate hnscc csc culture systems and refining techniques for the selection of well - defined cell populations from clinical samples. furthermore, major signaling pathways in csc or csc - like populations of hnscc are under investigation. the major cellular signaling mediators should be ideal targets for the development of new therapeutic agents to specifically eradicate high - risk hnscc cells, which may also hold drug - resistant phenotypes.	human head and neck cancer (hnc) is a highly heterogeneous disease. understanding the biology of hnc progression is necessary for the development of novel approaches to its prevention, early detection, and treatment. a current evolutional progression model has limitations in explaining the heterogeneity observed in a single tumor nest. accumulating evidence supports the existence of cancer stem cells (cscs) as small subpopulations in solid tumors, including hnc. these cscs can be selected by appropriate cell surface markers, which are cancer type specific and have been confirmed by unique in vitro and in vivo assays. selected csc populations maintain a self - renewal capability and show aggressive behaviors, such as chemoresistance and metastasis. in addition to introducing the csc concept in solid tumors, this short review summarizes current publications in hnc csc and the prospective development and application of the csc concept to hnc in the clinic.
breast cancer is the most common female malignancy and the second most common cause of death from cancer among white and black women. it is a worldwide main health problem linked with high levels of morbidity and mortality in developing countries due to delayed presentation. breast cancer affects more than one million females annually, the incidence increases with adoption of western life styles. the american cancer society in 2015 reported an estimated number of new cases of breast cancer in women in the united states to be 231,840 with an estimated death of 40,290. in nigeria, lack of public awareness of breast cancer and screening in the environment, absence of organized screening programs, lack of accessible and effective treatment options, and more importantly the cultural belief had made women to have late detection and presentations. early detection involves two major components which are education (to encourage early diagnosis) and screening. a wide range of modern procedures available for the screening includes mammography, sonography and magnetic resonance imaging of the breast. self breast examination (sbe) and clinical breast examination (cbe) are other methods that can be used to detect any changes in the breast. sbe involves the woman herself looking at and feeling the breasts for lumps, shape, texture, size and contour. the purpose of this is for any woman to be taught the topography of her breasts, know how her normal breasts feel and be able to identify changes in them should they occur in the future. it has been reported that the sensitivity and specificity values of sbe are difficult to determine, but it has a positive effect on the early detection of breast cancer. sbe has the advantages that it is a simple, inexpensive, non - invasive procedure which helps a woman to know her breast and allows her to detect any changes. recent studies no longer recommend the use of sbe for breast cancer screening as it did in the past because of the disadvantages of breast frequent healthcare visits, increased number of benign biopsy results, with increased biopsy leading to a higher risk of breast cancer and increased healthcare costs. the term breast awareness is now used to describe a woman s familiarity with her breasts and it has been suggested that periodic consistent sbe may facilitate this awareness. studies of community samples of diverse groups of women in the usa and canada show that the rates for performing monthly sbe ranged from 29 to 63%. sbe is the recommended general approach to increasing breast health awareness and thus potentially allow for early detection of any anomalies. although, the efficacy of sbe is debatable in countries where cbe and mammography are readily available, accessible and affordable, but where there is no facility for such, sbe remains a cost - effective method to detect breast changes. it has also been noted that despite the advent of modern screening methods, more than 90% of cases of breast masses are detected by women themselves, stressing the importance of sbe. a woman who performs sbe regularly and correctly is more motivated to seek medical attention, including cbe and mammography when need arises. in a study conducted among nurses in primary health care in ibadan, 80.9% acknowledged that early detection of breast cancer was through sbe but only 40.8% of them had the knowledge of the correct time that sbe should be performed monthly. this was however not too different from another study conducted also among the medical students in lagos, where 65.4% of students showed sbe awareness, 53.6% had the knowledge that both male and female are required to perform it. in the same study, it was reported that 23.8% of students had the knowledge that sbe should be performed daily, 22.5% weekly, 50.8% monthly and 2.9% yearly. of those that have never performed sbe among them, 46.7% did not because they did not have any symptoms, 26.7% felt it was not important while 22.6% did not know how to do it. however, among market women in abakaliki, nigeria, obaji and colleagues reported that 38.9% of women had heard of sbe, but 0.4% practiced it monthly. this study showed that there was association between the level of education and sbe awareness. amongst 231 female traders recruited during a cross - sectional study in ibadan, nigeria, only 37.1% were aware of sbe and 18.1% of the respondents had ever practiced sbe. another descriptive hospital - based study was carried out amongst employees of two main health institutions in bayelsa, nigeria. twenty - two (23.9%) practiced sbe once a month and only 3% practiced sbe more than once a month. these show a wide variation in the level of awareness and practice of sbe among different socio - economical and cultural status. it was a descriptive, cross sectional hospital - based study of the awareness of signs of breast cancer and practice of sbe among nurses working in a rural federal teaching hospital in nigeria (fethi). the study was conducted among the nurses in the departments of family medicine, obstetrics and gynecology, internal medicine, surgery, pediatrics, psychiatry, otorhinolaryngology and community health of fethi. these are the departments where postgraduate medical residency training is being run aside from accident and emergency unit and dental department. the study was conducted over a period of 8 weeks from february 1 to april 30, 2015. fethi is a referral tertiary health institution serving ekiti state in southwest nigeria and other adjoining towns of other states. the hospital metamorphosed in 1998 to federal medical centre from the then state owned general hospital, which was established in 1948. the federal tertiary institution is situated in ido - osi local government area with estimated population of 159,114 and that of ido ekiti town was 67,470 according to 2006 national population census. the hospital is currently accredited for postgraduate training in eight clinical fields and it has 250 nurses working in the various departments excluding accident and emergency and dental departments. consenting female nurses ages 20 to 60 years who were staff of fethi at the time of data collection participated in the study. a convenience sample of 90 nurses was recruited ; this was a derivative of calculated one third of 250 nurses present in the aforementioned eight departments, with an attrition of 10%. the questionnaire used was a modified and adapted version of the breast cancer awareness measure. this was pretested on 20 nurses in a comprehensive health center in ido - ekiti community who were not part of the sample size. the nurses were reached through the heads of the nursing units of each department and one department was sampled per week. the nurses who met the inclusion criteria in each department were subjected to simple randomization by picking yes or no on folded papers. the nurses who picked yes were made to fill the modified, standardized and pretested self administered semi structured questionnaires which were only numbered, while the researchers returned for collection from each nurse after 30 minutes. in this study there were 11 questions on the knowledge of close warning signs of breast cancer with answer options of yes, no, i do nt know. yes answer was apportion one (1) mark while no and i do nt know were scored 0 (zero). a total of 11 marks were obtainable, those who scored marks of 6 and above (> 50%) were said to have good knowledge of warning signs of breast cancer. seven questions were asked on knowledge of risk factors of breast cancer, the questions ranged from at what age can breast cancer occur, can overweight, relative with breast cancer, having children late in life, early menarche, late menopause and exercise be risk factors ? these were scored on likert scale and those nurses who agreed to four (> 50%) out of the seven stated risk factors were judged to have good knowledge of risk factor and less than that were ascribed poor knowledge. the knowledge of warning signs of breast cancer and knowledge of risk factors were considered together and nurses who scored 50% and above in both were said to have good knowledge of breast cancer. the nurses who did monthly sbe were ascribed regular performer (adherent) of sbe but those who were doing sbe occasionally, weekly or once every six months were tagged irregular performers (non - adherent). ethical approval was obtained from the research and ethical committee of fethi, ekiti state, nigeria. statistical analysis was done using computer software : statistical package for social sciences version 16.0 (ibm, usa version 16). ninety nurses were recruited for the study, but 85 questionnaires were valid for analysis. the age group of respondents ranged between 20 - 60 years, the predominant 37 (43.5%) age group was age 30 - 39 years, followed by age group 20 - 29, which was 23.5%. respondents knowledge of warning signs of breast cancer was assessed with the under listed eleven questions. breast lump and lump in the armpit were the most recognized warning signs of breast cancer closely followed by change in the shape of the breast or nipple (table 1). table 2 revealed that ever had a relative with breast cancer (51.8%) and women of any age (56.5%) were considered as risk factors for breast cancer, while overweight, late age at childbirth etc. figure 1 shows that about one - third, 27 (31.8%) of the respondents not only practiced self breast examination on monthly basis but were confident on how to do sbe while majority (68.2%) were not aware of monthly sbe and not confident on how to do sbe. majority of the respondents (87.1%) have never had cbe and 71.8% reported of prompt doctor s consultation if they found a change in their breasts. table 3 shows that majority (64.7%) of the nurses had good knowledge of the warning signs of breast cancer while majority (81.2%) had poor knowledge of risk factors of breast cancer. the knowledge of the breast cancer was generally poor among the nurses (84.7%) and majority of them did not know the correct time of performing sbe (68.2%). table 4 shows that the knowledge of breast cancer may not influence the practice sbe, p=0.6. ninety nurses were recruited for the study, but 85 questionnaires were valid for analysis. the age group of respondents ranged between 20 - 60 years, the predominant 37 (43.5%) age group was age 30 - 39 years, followed by age group 20 - 29, which was 23.5%. respondents knowledge of warning signs of breast cancer was assessed with the under listed eleven questions. breast lump and lump in the armpit were the most recognized warning signs of breast cancer closely followed by change in the shape of the breast or nipple (table 1). table 2 revealed that ever had a relative with breast cancer (51.8%) and women of any age (56.5%) were considered as risk factors for breast cancer, while overweight, late age at childbirth etc. figure 1 shows that about one - third, 27 (31.8%) of the respondents not only practiced self breast examination on monthly basis but were confident on how to do sbe while majority (68.2%) were not aware of monthly sbe and not confident on how to do sbe. majority of the respondents (87.1%) have never had cbe and 71.8% reported of prompt doctor s consultation if they found a change in their breasts. table 3 shows that majority (64.7%) of the nurses had good knowledge of the warning signs of breast cancer while majority (81.2%) had poor knowledge of risk factors of breast cancer. the knowledge of the breast cancer was generally poor among the nurses (84.7%) and majority of them did not know the correct time of performing sbe (68.2%). table 4 shows that the knowledge of breast cancer may not influence the practice sbe, p=0.6. more than half (64.7%) of the nurses in this study had good knowledge of the warning signs of breast cancer while only 18.8% had good knowledge of the risk factors of breast cancer. when the knowledge of warning signs and risk factors of breast cancers were put together, only 15.3% of the respondents had good knowledge of breast cancers. their knowledge of breast cancer was lower than the study conducted in ibadan among primary health care nurses and among egyptian nurses, which reported 60.9 and 39.9% respectively using the same methodology. in this study, only few of them were aware that early menarche (23.5%), late menopause (15.4%), late age at child birth (24.7%) were risk factors for breast cancer. poor knowledge of breast cancer risk factors had also been reported in a study among female healthcare professionals lagos, nigeria. the risk factors mostly identified in the study were increasing age (89%) and current use of oral contraceptive pills as breast cancer risk factor (82%). other risk factors such as family history of breast cancer, early age at menarche and late age at menopause were recognized by less than three - quarter of participants. least recognized risk factors were nulliparity and advanced age at first childbirth, while the least recognized risk factor in this study was late menopause followed by lack of exercise. this abysmal level of ignorance about risk factors and common symptoms of breast cancer in nigerian women generally had been reported in other literatures. a study has shown that having knowledge of breast cancer risk factors could be related to profession. ibrahim in his study reported 74% as doctors mean knowledge score for breast cancer, 35% for nurses while knowledge score in other allied professionals was 31%. difference in knowledge score between doctors and nurses was statistically significant (p<0.001), the study also showed that there was no statistically significant difference in knowledge score among nurses and other allied healthcare providers (p=0.6). similar results of poor knowledge of breast cancer was also estimated among nurses in the university hospital of rabat, morocco where only 43% had good knowledge of breast cancer risk factors, pakistan where the level of good knowledge of breast cancer risk factors among nurses working in teaching hospitals of karachi was 35%. though, these are higher than the 18.8% of knowledge of risk factor in our study, but they are generally considered poor being lower than expected average knowledge score for nurses. this study also observed that the practice of sbe examination among the nurses was poor. only a third (31.8%) of them practiced monthly sbe this observation was similar to what was reported in a hospital based study in bayelsa state, in nigeria where only 23.9% of nurses examined their breasts on monthly basis. only 40.8% of the nurses in ibadan study had the knowledge of the correct time that sbe should be performed monthly. similar reports of low practice of monthly sbe have been reported among women in general. this could be due to lack of knowledge on how to do breast self examination and some were not aware that sbe is done monthly. therefore, this may explain why knowledge of breast cancer was not significantly associated with practice of sbe in this study contrary to a study in malasia. perhaps the nurses in this study are aware of the current knowledge on sbe as no longer a screening method, this may contribute to their low practice of sbe. this study pointed out the gaps in the knowledge and awareness of breast cancer warning signs, risk factors, and practice of sbe among the nurses. this could be due to lack of adequate re - education and re - training of the nurses on breast cancer in our hospital. opportunity should therefore be sought in various health facilities to educate nurses who are supposed to be closer to patients on breast cancer, its risk factors, symptoms and warning signs. regular training and re - training on sbe and how practice of sbe on monthly basis as a readily available and at no cost a means of picking any changes in the breast with prompt presentation to hospital for further evaluation can be so helpful. one limitation of this study is that the nurses could not be assessed on their ability to practically perform sbe. our hospital has education committee like many tertiary health institutions in nigeria ; this study thereby reveals an area of gap in knowledge where attention needs to be focused as soon as possible in the hospital education committee s curriculum. our hospital has education committee like many tertiary health institutions in nigeria ; this study thereby reveals an area of gap in knowledge where attention needs to be focused as soon as possible in the hospital education committee s curriculum.	breast cancer is the most common female malignancy linked with high levels of morbidity and mortality in developing countries due to delayed diagnosis. this research assessed the knowledge of signs and risk factors of breast cancer and practice of self breast examination (sbe) among female nurses in a rural tertiary hospital. eighty - five nurses ages 20 to 60 years were sampled by simple randomization over a period of eight weeks through a self - administered semi - structured questionnaire. the analysis was done using statistical package for social science version 17. sixteen (15.3%) nurses had adequate knowledge of breast cancer, having a relative with breast cancer (51.8%) and a woman of any age (56.5%) were recognized by majority as risk factors for breast cancer. majority (68.2%) were not practicing monthly sbe and not confident on how to do it. this study pointed out the gaps in the knowledge and awareness of breast cancer and practice of sbe among the nurses. opportunity should therefore be sought in various health facilities to educate nurses who are supposed to be closer to the patients.
the epstein - barr virus (ebv) is a member of the herpesviridae virus family. it is estimated that the incidence of ebv infection among adults is between 90% and 95%. however, it is evident that ebv may cause infectious mononucleosis (i m) in the lytic phase of ebv s life cycle. past ebv infection is associated with burkitt lymphoma, hodgkin disease, and nasopharyngeal or stomach cancer. in addition, there is a hypothesis that past ebv infection can also lead to certain allergic and autoimmune diseases. once a person has been infected with ebv, she or he will carry ebv latent infection of b - lymphocytes. viral load remains relatively constant over time in circulating b - cells with a non - activated phenotype. although potential mechanisms of autoimmune diseases have not been clearly elucidated, both genetic and environmental factors, such as infectious agents, are considered to be responsible for their development. also, five possible mechanisms for the pathogenesis of autoimmune diseases have been distinguished : molecular mimicry activation of autoreactive t cells by microbial peptides with structural similarity to self - peptides ; viral and bacterial superantigens activation of autoreactive t cells that express particular v segments ; enhanced processing and presentation of auto - antigens by antigen - presenting cells recruited to an inflammatory site and followed by autoreactive lymphocyte priming ; bystander activation enhanced cytokine production that induces the expansion of autoreactive t cells ; activation of lymphocytes by lymphotropic viruses an infection of b cells resulting in b cell proliferation, excess antibody production, and formation of circulating immune complexes [6, 7 ]. epstein - barr virus is considered to be an aetiological factor of autoimmune diseases because of the following : the virus is a common pathogen responsible for the worldwide prevalence of autoimmune diseases ; ebv stays in the body throughout life, which explains the chronic course of autoimmune diseases that are often accompanied by exacerbations of symptoms ; the virus modifies the host immune response. it encodes a homolog of the bcl-2 oncogene that inhibits apoptosis and interferon (ifn-) signaling in b - cells. it is also responsible for changes in the production of pro - inflammatory cytokines, such as tumor necrosis factor (tnf-), interleukin (il)-1, and il-6, as well as of viral cytokines that share immunosuppressive properties with il-10 [8, 9 ]. interleukin 1, tnf-, and ifn- can induce hla class ii expression and lead to auto - antigen presentation and auto - reactive t - cell activation. the epstein - barr virus can also infect t lymphocytes. in 1971, evan demonstrated raised antibody titres to ebv in patients with systemic lupus erythematosus (sle). this was the reason to suspect that the ebv might participate in the development of autoimmune diseases. moreover, there is now evidence that ebv infection can cause autoimmune diseases, for example sle [13, 14 ], multiple sclerosis (ms), rheumatoid arthritis (ra), sjgren s syndrome [17, 18 ], or autoimmune hepatitis. several mechanisms of autoimmunity have been shown to be involved in the pathogenesis of these diseases. as far as lupus erythematosus is concerned, these are : high ebv dna viral loads, elevated ebv antibody concentrations, impaired ebv - specific t - cell responses, and the phenomenon of molecular mimicry [5, 20 ]. for rheumatoid arthritis, they include : high anti - ebv titres, cell - mediated control of ebv, cross - reactivity between ebv and human self - proteins (molecular mimicry), presence of the ebv genome in synovial membrane, and a cell - mediated response to the ebv within the joint. for primary sjgren s syndrome (pss), they are : more frequent presence of ebv dna in saliva and salivary tissue, as well as an elevated level of anti - ebv titres. patients with infectious mononucleosis have been reported with various antibodies in their serum. this can also suggest that there is a link between the ebv infection and autoimmune diseases. autoimmune thyroid disorders (aitds) are the most common organ - specific autoimmune diseases. autoimmune thyroid disorders develop upon activation of specific helper t cells directed against the thyroid antigens, the thyroid peroxidase (tpo), and the thyrotropin receptor (tshr). activated helper t cells induce b cells to secrete thyroid antibodies, such as the thyroid peroxidase antibodies (tpoabs) and the thyrotropin receptor antibodies (trabs). family clustering of these disorders may provide evidence for their genetic and environmental aetiology. according to hansen, environmental factors are also said to play a role in aitds, accounting for 20 - 40% of cases with regard to monozygous twins. other factors that are also considered to be responsible for autoimmune thyroid disorders include : irradiation, treatment with radioiodine, iodine or iodine - rich amiodarone, selenium intake, hormones, oral contraceptives, drugs, antiretrovirals, pregnancy and/or parity, stress, direct trauma, seasonal variation, smoking, campath-1h anti - cd52, ifn-, il-2, granulocyte - macrophage colony - stimulating factor (gm - csf), viral and/or bacterial infections, and lack of infections (the hygiene hypothesis) [27, 30 ]. this confirms an infectious aetiology of the disorders. to be specific, thyrocytes express a functional toll - like receptor 3 (tlr3) which, when overexpressed, may cause hashimoto s thyroiditis (ht) that can be induced by a virus infection. penhale and young have demonstrated that rats that have undergone both thymectomy and irradiation are less susceptible to aitds if maintained under specific pathogen - free conditions. in 1983, carter and smith infected chicken embryos with an avian leukosis virus, rav-7. as a result, tozolli, in turn, has suggested that toxoplasma gondii may participate in aitds. in a study by wasserman, it was shown that prior infection with toxoplasma gondii can be closely associated with elevated thyroid peroxidase antibodies. in addition, researchers have shown that htlv-1 may have an impact on the development of both hashimoto s thyroiditis [37, 38 ] and graves disease. other studies have found that there is a possible link between hiv infection and autoimmune thyroid disorders [40, 41 ]. also, human foamy virus (hfv) proteins have been detected in the thyroid tissue of patients with graves disease. serological data indicates that the influenza virus, hepatitis c virus, enterobacteriaceae, streptococci, staphylococci, yersinia, and helicobacter can have an influence on aitds as well [26, 42 ]. nevertheless, conflicting data has also been published with regard to the rubella virus, the parvovirus, as well as hepatitis b and c viruses. in one study, thyroid tissue specimens obtained from aitd patients and patients with multinodular goitre have been investigated to detect herpesviridae dna. the group of aitd patients have been reported with herpesviridae dna more frequently than the other group. however, there was no statistical significance observed in aitd patients, nor in patients with any other viruses. there is a hypothesis that in genetically susceptible patients, ebv - infected autoreactive b - cells seed the thyroid gland, produce autoantibodies, and send co - stimulatory signals to autoreactive t - cells. normally, the ebv infection is kept under control, especially by cytotoxic cd8 + t - cells that eliminate proliferating and lytically infected b - cells. impaired ebv control may result from a decreased number of ebv - specific cd8 + t - cells. an increased cd4/cd8 ratio is characteristic of autoimmune diseases. in their study, akahori. presented three cases of patients suffering from graves disease comorbid with infectious mononucleosis due to primary ebv infection. they suggested that inflammation from viral infection might be associated with the development of graves disease. nagata has shown that in vitro reactivation of the ebv infection causes the production of thyrotropin receptor antibodies (trabs) in ebv - infected b - cells with trabs on their surface. moreover, nagata has also reported increased trab titres in children with infectious mononucleosis due to ebv primary infection. in a study by janegova, subjects with hashimoto s thyroiditis were reported with latent membrane protein 1 (lmp1), which was not revealed in the case of graves disease patients. epstein - barr virus - encoded small rnas (ebers) were detected in both hashimoto s thyroiditis and graves disease patients, exclusive of negative control samples. in addition, an elevated serum level of epstein - barr nuclear antigen (ebna) was observed in patients with hashimoto s thyroiditis. also, it was found that antibodies against ebv viral capsid antigen (igg - vca) and against early antigen (igg - ea - d / dr) were more common for patients with thyroiditis versus the controls. the seroprevalence of ebv infection was reported to be higher in children with autoimmune thyroid disorders when compared to the controls. however, tozzoli failed to observe elevated ebv - igg levels in aitd patients when compared to the healthy controls. it is said that thyroid autoantibodies occur more frequently in subjects with autoimmune diseases versus the general population. experts suggest that thyroid function screening should be conducted in patients with primary sjgren s syndrome, rheumatoid arthritis, and lupus erythematosus because these diseases may be connected with ebv infection. the ebv can lead to in vitro transformation of normal resting b lymphocytes to proliferating lymphoblasts. moreover, the virus can be found in many lymphomas therefore, researchers suggest that ebv may participate in the malignant transformation of hashimoto s disease into malignant lymphoma of the thyroid (table 1). over 90% of all primary thyroid lymphomas are diagnosed in individuals with a history of hashimoto s disease, and the majority of them are of b - cell origin. a possible link between the epstein - barr virus (ebv) infection and autoimmune thyroid disorders (aitd) according to literature based on the study results, ebv is not the only agent responsible for the development of autoimmune thyroid diseases. further investigations still need to be undertaken to explain the link between the ebv infection and autoimmune thyroid disorders.	the epstein - barr virus (ebv), also known as human herpesvirus 4, is a member of the herpesviridae virus family. ebv infection can cause infectious mononucleosis (i m) in the lytic phase of ebv s life cycle. past ebv infection is associated with lymphomas, and may also result in certain allergic and autoimmune diseases. although potential mechanisms of autoimmune diseases have not been clearly elucidated, both genetic and environmental factors, such as infectious agents, are considered to be responsible for their development. in addition, ebv modifies the host immune response. the worldwide prevalence of autoimmune diseases shows how common this pathogen is. normally, the virus stays in the body and remains dormant throughout life. however, this is not always the case, and a serious ebv - related illness may develop later in life. this explains the chronic course of autoimmune diseases that is often accompanied by exacerbations of symptoms. based on the present studies, ebv infection can cause autoimmune diseases, such as systemic lupus erythematosus (sle), multiple sclerosis (ms), rheumatoid arthritis (ra), sjgren s syndrome, and autoimmune hepatitis. the ebv has also been reported in patients with autoimmune thyroid disorders. although ebv is not the only agent responsible for the development of autoimmune thyroid diseases, it can be considered a contributory factor.
	noble metals, especially gold, have been widely used in plasmon resonance applications. although silver has a larger optical cross section and lower cost than gold, it has attracted much less attention because of its easy corrosion, thereby degrading plasmonic signals and limiting its applications. to circumvent this problem, we report the facile synthesis of superstable agcu@graphene (acg) nanoparticles (nps). the growth of several layers of graphene onto the surface of agcu alloy nps effectively protects the ag surface from contamination, even in the presence of hydrogen peroxide, hydrogen sulfide, and nitric acid. the acg nps have been utilized to enhance the unique raman signals from the graphitic shell, making acg an ideal candidate for cell labeling, rapid raman imaging, and sers detection. acg is further functionalized with alkyne - polyethylene glycol, which has strong raman vibrations in the raman - silent region of the cell, leading to more accurate colocalization inside cells. in sum, this work provides a simple approach to fabricate corrosion - resistant, water - soluble, and graphene - protected agcu nps having a strong surface plasmon resonance effect suitable for sensing and imaging.
the disease caused by autoantibodies with specificity for the nc1 domain of the 3-chain of type iv collagen, often referred to as anti - glomerular basement membrane disease (anti - gbm disease) or goodpasture 's disease, is characterized by rapidly progressive glomerulonephritis (gn) often accompanied by lung haemorrhage. disease progression is usually dramatic, and a substantial proportion of patients in recently published series present with oliguric or anuric acute renal failure [24 ]. detectable amounts of anti - gbm antibodies usually do not remain in the circulation for > 612 months, even without the use of cytotoxic drugs. after that time, the recurrence of anti - gbm antibodies as well as relapse of glomerulonephritis (gn) is rare [35 ]. here, we report a case of a clinically and histologically mild form of gn, associated with low levels of anti - gbm antibodies but complicated by a protracted relapsing course. a 71-year - old woman with a medical history that included a peripheral facial paresis and an irritable colon sought medical attention in august 2000 after a 6-month period of general malaise, intermittent subfebrility and myalgia. blood tests indicated a mild renal failure with a serum creatinine of 100 mol / l and an erythrocyte sedimentation rate of 80 mm / h. a week later her creatinine had risen to 130 mol / l and serology was positive for anti - gbm with 61 and myeloperoxidase - antineutrophil cytoplasmic anitbody (mpo - anca) > 320 elisa units (above 10 is considered as positive in both the assays). she was transferred to the lund university hospital, where she received apheresis with protein a adsorption columns. a renal biopsy showed focal necrotizing glomerulonephritis with crescents in 6 of 16 glomeruli and a typical linear immunofluorescence pattern for igg, kappa and lambda. after 5 months, the medication was switched to azathioprine 50 mg daily, which was continued for 12 months. serum creatinine remained stable at a level of 90 mol / l and anti - gbm antibodies were below the detection limit (figure 1). 1.the solid line represents levels of anti - gbm antibodies measured by the enzyme - linked immunosorbent assay (elisa) and expressed in arbitrary elisa units as indicated by the right vertical axis. the serological relapses coincided with clinical relapses ; start (and restart) of immunosuppressive treatment is indicated by arrows. method was used until june 2007, then method was used until november the same year when method, using international units, was introduced. elisa units for mpo - anca are indicated on the left vertical axis, levels differ between the three assays. the solid line represents levels of anti - gbm antibodies measured by the enzyme - linked immunosorbent assay (elisa) and expressed in arbitrary elisa units as indicated by the right vertical axis. the serological relapses coincided with clinical relapses ; start (and restart) of immunosuppressive treatment is indicated by arrows. method was used until june 2007, then method was used until november the same year when method, using international units, was introduced. elisa units for mpo - anca are indicated on the left vertical axis, levels differ between the three assays. two years later in the fall of 2004, she experienced a relapse with re - emergence of anti - gbm antibodies, return of microscopic haematuria and a slight elevation of serum creatinine (maximum 108 mol / l). anti - gbm remained positive at low level, but turned negative in april 2006., she experienced her second minor relapse with return of anti - gbm antibodies, microscopic haematuria and a slight elevation of creatinine (maximum 131 mol / l). treatment with pulse cyclophosphamide therapy was initiated, resulting in disappearance of anti - gbm antibodies and haematuria., both haematuria and anti - gbm antibodies returned, like at previous relapses there was no malaise or other symptoms of general inflammation. this third relapse was curbed with an increase in azathioprine doses to 150 mg and reinstitution of a moderate prednisolone dose. once again, there was a response with vanishing haematuria and autoantibodies. at the most recent follow - up in october 2011, anti - gbm was negative, there was no haematuria, and creatinine was 106 mol / l. the clinical course in this case was benign, with preserved renal function for more than a decade. most cases of anti - gbm disease are diagnosed at a more advanced stage. in our series of 75 cases, it is impossible to discern whether the benign clinical course was due to the inherent nature of the case or a direct result of the swift initiation of aggressive autoantibody lowering therapy. a finding in favour of a mild inherent nature in this case is the relatively low levels of anti - gbm antibodies, 61 elisa units, compared with a median level of anti - gbm antibodies in positive cases at the laboratory of 112 units (interquartile range 39197). previous studies have indicated that levels of anti - gbm antibodies at diagnosis are predicative of long - term outcome [2, 3 ]. however, it is probable that without treatment, levels of anti - gbm would have increased along with an accelerated intensity of the glomerulonephritis. the most remarkable feature of this case is the three mild relapses. in all instances, the return of anti - gbm antibodies was associated with the return of microscopic haematuria, strongly indicating a pathogenic capacity of the autoantibodies. however, there are only a few reports of late relapses of anti - gbm disease [57 ]. reported two cases with relapses among 71 patients in his long - term follow - up study and we found one among 75. in the present case, this combination is common and was found in 30% of the patients in our swedish cohort, and other studies have found similar frequencies. the mpo - anca levels fluctuated, and even though four peaks can be identified, the mpo - anca and anti - gbm curves were not parallel. a temporal separation of anti - gbm and mpo - anca in the form a sequential appearance of the autoantibodies has previously been reported [9, 10 ]. the combination of anca and anti - gbm could indicate some sort of overlap syndrome between goodpasture 's disease and anca - associated vasculitis, but while there was a strict correlation between anti - gbm and microscopic haematuria, there was no obvious correlation between the mpo - anca levels and clinical findings. also in the case described by serratrice., the flare of anti - gbm disease was preceded by a rise in mpo - anca levels that had peaked several months earlier. in summary, this case shows that anti - gbm antibodies might be present in cases with relatively mild renal failure, and that high vigilance is necessary as mild disease might be at hand only during a relatively short window of opportunity. even though this case might represent an overlap syndrome with anca - associated vasculitis, the return of the anti - gbm antibodies several years after successful treatment demonstrates that long - term surveillance is warranted. how the follow - up should be organized with respect to frequency of visits and anti - gbm test however, the present case suggests that negative dipstick tests for haematuria might be sufficient to rule out recurrence of anti - gbm nephritis.	anti - glomerular basement membrane disease (anti - gbm) is usually characterized by rapidly progressive glomerulonephritis, and when autoantibody production has ceased, relapses are rare. here, we report a 71-year - old women diagnosed at a stage of mild renal insufficiency. over a period of 10 years, she experienced three mild relapses with return of anti - gbm antibodies, haematuria and slight elevations in serum creatinine level. all three relapses responded to immunosuppressive therapy, and all were preceded by peaks of myeloperoxidase - antineutrophil cytoplasm antibodies (mpo - anca). this case shows that long - term follow - up is warranted in patients treated for anti - gbm - mediated disease, but urinary dipsticks may be sufficient for early detection of relapses.
the porphyrias comprise a heterogeneous group of rare, mainly hereditary, diseases caused by partial deficiencies in one of the eight enzymes involved in the heme biosynthesis (puy. symptoms of disease can present as acute attacks of abdominal pain and neuropsychiatric symptoms [delta aminolevulinic acid dehydratase (alad) deficiency and acute intermittent porphyria (aip) ], cutaneous symptoms [congenital erythropoietic porphyria (cep), porphyria cutanea tarda (pct), and erythropoietic protoporphyria (epp) ] or both (hereditary coproporphyria (hcp) and variegate porphyria (vp)). acute attacks are triggered by various factors, such as endocrine changes, physical or emotional stress, alcohol consumption, smoking, and a wide array of drugs. cutaneous symptoms consist of blistering and fragile skin on sun - exposed areas (cep, pct, hcp, vp) and photosensitization (epp). with the exception of pct, which also occurs in a sporadic, nonhereditary form, most porphyrias are inherited in an autosomal dominant fashion with low clinical penetrance. as with many other rare diseases, little pregnancy has been reported to exacerbate or improve cutaneous symptoms in women with pct (baxi. 1983 ; loret de mola. 1996 ; aziz ibrahim and esen 2004) and epp (bewley. 1998 ; jacquemyn 2003 ; madu and whittaker 2006), and women with aip sometimes have their first acute attack during pregnancy (engelhardt. however, how porphyria can affect pregnancy outcomes has seldom been investigated, and the body of evidence only consists of case reports and small case series. acute porphyrias have been implied to be risk factors for low birth weight, growth retardation, premature delivery, spontaneous abortion, and perinatal death (brodie. 1977 ; olund 1988 ; kanaan. 1998 ; aggarwal. 2002 ; andersson. 2003 ; muralidhar. 2006 ; cappell 2008), whereas pct has been suggested to increase the risk of preeclampsia (loret de mola. 1996 the aim of this study was to assess whether cutaneous or acute porphyria has been associated with excess risks of adverse pregnancy outcomes, specifically preeclampsia, delivery by caesarean section, low birth weight, premature delivery, small for gestational age (sga) infants, perinatal death, and congenital malformations. two national registries, the norwegian porphyria register and the medical birth registry of norway (mbrn), provided the basis of a large - scale population - based cohort study to address these issues. the norwegian porphyria centre (napos) has a national responsibility for diagnosing porphyric disease in norway. all norwegian porphyria patients, both with active disease and those presymptomatically examined (latent porphyria) are invited to participate in the norwegian porphyria register, established in 2002 and based on written consent. approximately 70% of all known porphyria patients in norway participate by filling in a disease - specific questionnaire at the time of diagnosis and a follow - up questionnaire every second year thereafter. in addition, laboratory data of porphyrin analyses are included whenever routine samples are sent for analyses. the register contains information on diagnosis, biochemical characteristics, symptoms, treatment, daily life activities, and quality of life. by september 2009, the mbrn is based on compulsory notification of all live births and stillbirths in the country from 16 weeks of gestation and from 2001 from 12 weeks of gestation (irgens 2000). the mbrn comprises demographic data on parents and child, maternal health before and during index pregnancy, interventions and complications during pregnancy and delivery, and the newborns condition. the mbrn is routinely linked to the central population registry to obtain data on death dates. during the autumn of 2009, all women older than 18 years in the norwegian porphyria register were sent a letter of information about our study. data on diagnosis, biochemical characteristics (highest recorded urinary excretion of ala and pbg in women with acute porphyrias (aip, hcp, or pv), highest recorded total urine porphyrins in women with pct, and highest recorded erythrocyte protoporphyrins in women with epp), disease status, and lifestyle parameters of 318 women with porphyria were obtained. enabled by each woman s unique national identification number, these data were linked with records of deliveries at the mbrn. data on year of delivery, maternal age, parity, preeclampsia, caesarean section, birth weight, gestational age, perinatal death (stillbirth at gestational age 16 weeks, or death during the first 7 days of life), and congenital malformations (any and serious, as defined by the mbrn) were obtained from the mbrn. sga, a proxy for in utero growth retardation, was defined as birth weight below the tenth percentile for actual gestational age (skjaerven and bakketeig 1989). the study population ultimately consisted of the 2,275,715 singletons born between 1967 and 2006 by 1,100,591 unique mothers. the exposed group consisted of the 398 singletons born by the 200 mothers with porphyria. biochemical data standardized by urine creatinine values were available for 180 of these mothers. in agreement with others (de siervi. 1999 ; kauppinen and von und zu fraunberg 2002), the 90 women with acute porphyria (aip, hcp, or vp) were categorized as having active or latent disease depending on whether or not they had reported symptoms that could be attributed to acute porphyria to the norwegian porphyria register. deliveries by women with pct were stratified according to whether a genetic cause of the disease had been established by demonstration of a mutation in the uroporphyrinogen decarboxylase gene (familial pct) or not (sporadic pct). the 22 deliveries by mothers with unknown pct status were excluded from further analyses. because of the small number of deliveries in the epp group, they were also excluded from further analyses. no patients with cep or alad deficiency were registered in the norwegian porphyria register. the mann risks of adverse pregnancy outcomes were compared between the porphyric mothers and the rest of the population using 2 2 tables and logistic regression models (stata intercooled, version 9). for rare outcomes, the effect - estimate odds ratio (or) was considered a sufficiently good approximation of the relative risk (rr). for the sga outcome, however, maternal age, parity, and year of delivery were considered possible confounders and hence adjusted for as categorical variables in the regression models (maternal age : < 25, 25 - 34, 35 + years, parity : nulliparous / multiparous, year of delivery : 1967 - 1976, 1977 - 1986, 1987 - 1996, 1997 - 2006). we checked for statistically significant interactions between exposure and each of the possible confounders ; none were found. as subsequent deliveries by the same mother are not independent incidents, robust variances, cis and p values were estimated using the cluster the 200 mothers with porphyria had 398 singleton deliveries between 1967 and 2006 (table 1). compared with controls, they were in general younger, had a higher parity, and a higher proportion of deliveries during the earlier parts of the study period. table 1singleton deliveries by women with porphyria and the rest of the population (controls). norway, 1967 - 2006acute intermittent porphyria (aip)hereditary coproporphyria (hcp)variegate porphyria (vp)erythropoietic protoporphyria (epp)porphyria cutanea tarda (pct)controlsno. mothers801981021,100,391characteristics of population maternal age in years (%) < 2536.650.042.96.735.133.0 25 - 3457.750.047.686.760.557.0 35 + 5.70.09.56.74.310.0 parity (%) nulliparous39.450.033.346.738.441.3 multiparous60.650.066.753.361.658.7 year of delivery (%) 1967 - 197626.3047.626.761.127.4 1977 - 198628.05038.126.721.122.2 1987 - 199629.1509.520.010.825.6 1997 - 200616.604.826.77.024.9no. adverse pregnancy outcomes pre - eclampsia / eclampsia2010566,211 caesarean section901310207,947 birth weight < 2500 g30231395,500missing data5335 gestational age < 37 weeks902212124,959 missing data1113121,552 small for gestational age2002431232,051 missing data1113126,028 perinatal death3011529,938 congenital malformation, any2110373,106 congenital malformation, serious1010347,375gestational age 16 weeks, stillborn or died during first seven days of lifeas defined by the medical birth registry of norway singleton deliveries by women with porphyria and the rest of the population (controls). norway, 1967 - 2006 gestational age 16 weeks, stillborn or died during first seven days of life as defined by the medical birth registry of norway women with active acute disease had higher recorded urinary excretion of ala (p = 0.013) and pbg (p = 0.001) compared with women with latent acute disease (table 2). there were no statistically significant differences between the highest recorded urinary excretions of total porphyrins among the subgroups with pct. all women with pct had a high urinary excretion of uro- and hepta porphyrins compared with coproporhyrins. table 2biochemical characteristics of 180 mothers with porphyriadiagnosisanalyte (unit)upper reference limitno.median (25 - 75 percentiles)latent acute porphyriaala (mol / mmol creatinine)<5.0254.2 (2.5 - 7.2)pbg (mol / mmol creatinine)<0.8232.1 (0.4 - 7.9)active acute porphyriaala (mol / mmol creatinine)<5.0596.7 (3.8 - 11.8)pbg (mol / mmol creatinine)<0.8587.2 (3.3 - 13.9)erythropoietic protoporphyriafree erythrocyte protoporphyrin (mol / l erythrocytes)<1.9719.0 (7.8 - 34.3)sporadic porphyria cutanea tardatotal urine porphyrins (nmol / mmol creatinine)<30371,028 (686 - 1467)familial porphyria cutanea tardatotal urine porphyrins (nmol / mmol creatinine)<3044825 (463 - 1350)unknown type porphyria cutanea tardatotal urine porphyrins (nmol / mmol creatinine)<3081,068 (600 - 2118)ala delta aminolevulinic acid, pbg porphobilinogenacute intermittent porphyria, hereditary coproporphyria, or variegate porphyria biochemical characteristics of 180 mothers with porphyria ala delta aminolevulinic acid, pbg porphobilinogen acute intermittent porphyria, hereditary coproporphyria, or variegate porphyria among the 136 deliveries in the group with active acute porphyria, the estimate of excess risk of perinatal death did not reach statistical significance (table 3). however, when the analyses were restricted to firstborn children only, an almost fivefold increased risk of perinatal death was observed [adjusted or 4.9 (1.5 - 16.0) ]. among the 95 deliveries in the familial pct group, a threefold increased risk of perinatal death all the women experiencing perinatal loss belonged to different families and carried private mutations (three in the hydroxymethylbilane synthase gene, one in the protoporphyrinogen oxidase gene, and four in the uroporphyrinogen decarboxylase gene), as is common in the porphyrias. table 3risk of selected adverse outcomes in pregnancies of women with latent and active acute porphyria in comparison with the rest of the population (controls). norway, 1967 - 2006outcomecontrols (n = 2,275,317)latent acute porphyria (n = 62)active acute porphyria (n = 136)per 100 deliveriesper 100 deliveriesor crude (95% ci)or adjusted (95% ci)per 100 deliveriesor crude (95% ci)or adjusted (95% ci)preeclampsia and/or eclampsia2.90.0 - -2.20.8 (0.2 - 3.3)0.8 (0.2 - 3.6)caesarean section9.14.80.5 (0.2 - 1.5)0.5 (0.2 - 1.6)5.10.5 (0.2 - 1.5)0.6 (0.2 - 1.7)birth weight < 2,500 g4.21.60.4 (0.1 - 2.7)0.4 (0.1 - 2.8)2.90.7 (0.3 - 1.9)0.7 (0.3 - 1.9)gestational age < 37 weeks5.80.0 - -8.71.6 (0.8 - 3.0)1.6 (0.8 - 3.1)small for gestational age10.89.80.9 (0.4 - 2.2)0.9 (0.4 - 2.2)12.71.2 (0.6 - 2.2)1.1 (0.6 - 2.1)perinatal death1.40.0 - -3.22.3 (0.9 - 6.3)2.3 (0.8 - 6.1)congenital malformation, any3.23.21.0 (0.2 - 4.1)1.0 (0.3 - 4.2)1.50.4 (0.1 - 1.8)0.5 (0.1 - 1.9)congenital malformation, serious2.13.21.6 (0.4 - 6.4)1.6 (0.4 - 6.5)0.0 - -or odds ratio, ci confidence intervaladjusted for year of delivery (1967 - 1976, 1977 - 1986, 1987 - 1996, 1997 - 2006), maternal age (< 25, 25 - 34, 35 + years) and birth order (nulliparous / multiparous)effect measure relative risk (rr)gestational age 16 weeks, stillborn or died during first 7 days of lifetable 4risk of selected adverse outcomes in pregnancies of women with sporadic and familial porphyria cutanea tarda in comparison with the rest of the population (controls). norway, 1967 - 2006outcomecontrols (n = 2,275,317)sporadic porphyria cutanea tarda (n = 68)familial porphyria cutanea tarda (n = 95)per 100 deliveriesper 100 deliveriesor crude (95% ci)or adjusted (95% ci)per 100 deliveriesor crude (95% ci)or adjusted (95% ci)preeclampsia and/or eclampsia2.90.0 - -5.31.9 (0.6 - 6.1)2.2 (0.7 - 7.4)caesarean section9.12.90.3 (0.1 - 1.2)0.6 (0.1 - 2.6)5.30.6 (0.2 - 1.6)0.8 (0.3 - 2.5)birth weight < 2,500 g4.28.82.2 (0.8 - 5.6)2.2 (0.8 - 5.6)5.31.3 (0.5 - 3.4)1.3 (0.5 - 3.5)gestational age < 37 weeks5.89.71.7 (0.8 - 3.8)1.8 (0.8 - 4.1)5.71.0 (0.4 - 2.6)1.0 (0.4 - 2.8)small for gestational age10.824.22.2 (1.3 - 3.7)2.0 (1.2 - 3.4)13.61.3 (0.7 - 2.4)1.2 (0.6 - 2.2)perinatal death1.41.61.1 (0.2 - 8.1)0.9 (0.1 - 6.7)4.53.4 (1.3 - 8.5)3.0 (1.2 - 7.7)congenital malformation, any3.21.50.4 (0.1 - 3.1)0.6 (0.1 - 4.3)2.10.6 (0.2 - 2.5)0.8 (0.2 - 3.0)congenital malformation, serious2.11.50.7 (0.1 - 4.9)0.9 (0.1 - 6.8)2.11.0 (0.3 - 3.9)1.2 (0.3 - 4.8)or odds ratio, ci confidence intervaladjusted for year of delivery (1967 - 1976, 1977 - 1986, 1987 - 1996, 1997 - 2006), maternal age (< 25, 25 - 34, 35 + years), and birth order (nulliparous / multiparous)effect measure relative risk (rr)gestational age 16 weeks, stillborn or died during first seven days of life risk of selected adverse outcomes in pregnancies of women with latent and active acute porphyria in comparison with the rest of the population (controls). norway, 1967 - 2006 or odds ratio, ci confidence interval adjusted for year of delivery (1967 - 1976, 1977 - 1986, 1987 - 1996, 1997 - 2006), maternal age (< 25, 25 - 34, 35 + years) and birth order (nulliparous / multiparous) effect measure relative risk (rr) gestational age 16 weeks, stillborn or died during first 7 days of life risk of selected adverse outcomes in pregnancies of women with sporadic and familial porphyria cutanea tarda in comparison with the rest of the population (controls). norway, 1967 - 2006 or odds ratio, ci confidence interval adjusted for year of delivery (1967 - 1976, 1977 - 1986, 1987 - 1996, 1997 - 2006), maternal age (< 25, 25 - 34, 35 + years), and birth order (nulliparous / multiparous) effect measure relative risk (rr) gestational age 16 weeks, stillborn or died during first seven days of life among the 68 deliveries in the sporadic pct group, a two - fold increased risk of sga was observed (table 4). when analyses were restricted to firstborn children only, there were also increased risks of low birth weight [adjusted or 3.4 (1.2 - 10.0) ] and premature delivery [adjusted or 3.5 (1.2 - 10.5) ]. no case of preeclampsia was observed among mothers with sporadic pct (table 4). for mothers with familial pct, a nonsignificant estimate of a twofold increased risk of preeclampsia was observed. restricting analyses to firstborn children first - time mothers with active acute porphyria and mothers with familial pct had excess risks of experiencing perinatal loss. mothers with sporadic pct had an excess risk of giving birth to sga infants, and first - time mothers with sporadic pct also had increased risks of low birth weight infants and premature delivery. for the remainder, and thus the majority of the adverse pregnancy outcomes studied, no excess risks were observed for porphyric mothers. pregnancy outcomes in porphyric women have previously not been examined in large - scale population - based cohort studies, and the application of this design is the greatest strength of this study. reporting to the mbrn is compulsory, and the degree of completeness is very high (irgens 2000). one weakness of the study, however, is the misclassification of an unknown number of mothers with porphyria as controls. porphyric mothers not participating in the norwegian porphyria register, as well as undiagnosed mothers, were included in the control group. this was probably particularly relevant for deliveries in the most recent time periods, during which mothers might not have been diagnosed by the time of data collection in 2009, especially mothers with pct, which usually occurs after childbearing age. however, as this misclassification would have been independent of the outcomes registered, it would have caused effect estimates to be biased toward the null value. the possibility of residual confounding is another potential weakness. in particular, important lifestyle parameters maternal educational level, a useful proxy of social status, and body mass index (bmi) are not recorded, and smoking habits were only recorded from 1999 onward. patients with acute porphyrias have to adhere to a healthy lifestyle to avoid inducing acute attacks. pct, however, is precipitated by factors such as iron overload, hepatitis, alcohol use, and estrogens. thus, lifestyle factors could be the cause of the association between pct and adverse pregnancy outcomes. in the largest study on porphyrias and pregnancy to date, the authors reviewed the obstetric histories of 50 women with active acute porphyria and their total of 129 pregnancies during the 1950s and 1960s (brodie. 1977). they observed an 8% frequency of perinatal death, whereas in our study, a frequency of 3.2% was found. in our cohort, this represented an excess risk of perinatal death compared with the general population. did not include a control group, their results can not be directly compared with ours. published case reports on four pregnancies by three women with active aip reported that all four infants were born sga and one died shortly after delivery (olund 1988 ; wenger. 2006) reported that of four pregnancies by a woman with vp, two infants were sga and two died perinatally. reported that babies born after an acute attack had occurred during pregnancy had lower birth weight than if no acute attack had occurred, but is not apparent whether this difference was due to in utero growth retardation or premature delivery. a publication bias toward pregnancies with adverse outcomes is to be expected in case reports. we did not observe an excess risk of sga for mothers with active acute porphyria. a review of six published case reports on pct and pregnancy reported that all had good perinatal outcomes, but two developed mild preeclampsia and one gestational diabetes (loret de mola. based on this report, others have also suggested an association between pct and preeclampsia (aziz ibrahim and esen 2004). the point estimates of excess risk suggested a twofold increased risk of preeclampsia in familial pct, without reaching statistical significance, and no excess risk in sporadic pct. to our knowledge, no one has previously demonstrated an association between sporadic pct and sga infants, and for first - time mothers, a significant excess risk of low birth weight and premature delivery. more than 24% of singletons born by mothers with sporadic pct had a weight below the tenth percentile for their actual gestational age, whereas no such excess risk was observed in familial pct. lifestyle - related factors such as alcohol use and hepatitis are, to a larger extent, associated with sporadic pct than is familial pct (aarsand. 2009). at the time of diagnosis, 75% of women in the sporadic pct group reported smoking daily or occasionally, whereas 22% in the familial pct group did the same. frequencies of inactive life style (less than 1 h physical activity per week) were 27% and 13% respectively. smoking during pregnancy is a well known risk factor of low birth weight and sga (reeves and bernstein 2008), and inactive lifestyle is associated with high bmi, which is a suggested risk factor of preterm delivery (torloni. therefore, a less healthy lifestyle among women with sporadic pct than those with familial pct might explain the observed differences in risk of low birth weight, preterm delivery, and sga. the opposite pattern was observed for perinatal death, of which mothers with familial pct, but not with sporadic pct, had an excess risk. pct has been associated with diabetes (bleasel and varigos 2000), which is a known risk factor for perinatal death (balsells. however, none of the women with pct were registered with pregestational or gestational diabetes in the mbrn, although complicating factors such as diabetes are, in general, underreported to some extent (engeland. a common or coinherited genetic cause of both familial pct and perinatal death could be an explanation, even though the four women with familial pct and perinatal loss all belonged to different families and harbored private mutations in the uroporphyrinogen decarboxylase gene. an increased risk of perinatal death was also observed for mothers with active acute porphyria but not for mothers with latent disease. active, symptomatic disease is characterized by increased plasma and urine levels of heme precursors during acute attacks, and as many as two thirds of patients with active aip have increased levels also when in remission (kauppinen and von und zu fraunberg 2002). however, though less common, patients with latent disease can also present with increased urine and plasma levels. the exact mechanism by which the heme precursors cause acute symptoms is not fully understood, but neurotoxicity has been suggested (pischik and kauppinen 2009). whether the precursors can cross the placental barrier and be harmful to the fetus, damage the placenta, or in some other way compromise the intrauterine environment, remains highly speculative. alternatively, treatment of porphyric attacks might be harmful, even if several recently published case reports have suggested the use of heme arginate during pregnancy is safe (marsden and rees 2010 ; badminton and deybach 2006). yet again, a common or coinherited genetic cause of active acute porphyria and perinatal loss might exist, even if the women had different mutations as the cause of their acute porphyria. in conclusion, in this population - based cohort study, first - time mothers with active acute porphyria and mothers with familial pct had excess risks of perinatal loss. however, the findings suggest that women with porphyria should be monitored closely during pregnancy.	the porphyrias comprise a heterogeneous group of rare, primarily hereditary, metabolic diseases caused by a partial deficiency in one of the eight enzymes involved in the heme biosynthesis. our aim was to assess whether acute or cutaneous porphyria has been associated with excess risks of adverse pregnancy outcomes. a population - based cohort study was designed by record linkage between the norwegian porphyria register, covering 70% of all known porphyria patients in norway, and the medical birth registry of norway, based on all births in norway during 19672006. the risks of the adverse pregnancy outcomes preeclampsia, delivery by caesarean section, low birth weight, premature delivery, small for gestational age (sga), perinatal death, and congenital malformations were compared between porphyric mothers and the rest of the population. the 200 mothers with porphyria had 398 singletons during the study period, whereas the 1,100,391 mothers without porphyria had 2,275,317 singletons. first - time mothers with active acute porphyria had an excess risk of perinatal death [adjusted odds ratio (or) 4.9, 95% confidence interval (ci) 1.516.0 ], as did mothers with the hereditable form of porphyria cutanea tarda (pct) (3.0, 1.27.7). sporadic pct was associated with an excess risk of sga [adjusted relative risk (rr) 2.0, 1.23.4 ], and for first - time mothers, low birth weight (adjusted or 3.4, 1.210.0) and premature delivery (3.5, 1.210.5) in addition. the findings suggest women with porphyria should be monitored closely during pregnancy.
twenty - three consecutive, previously untreated patients with acute unilateral on were recruited from the ophthalmology outpatient clinics of isfahan university of medical sciences, iran between october 2009 and february 2010. these included four men and 19 women, with a mean (standard deviation [sd ]) age of 27.2 (7.0) years, ranging from 19 to 47 years. the inclusion criterion was typical clinical presentation of acute unilateral on before the age of 50 years and exclusion criteria were bilateral on, recurrent on and any disease or anomaly of the contralateral eye. no patient had a history of any major systemic diseases, including cardiovascular disease, arterial hypertension, hyperlipidemia or diabetes mellitus. all patients underwent neurologic and ophthalmologic examinations including visual acuity assessment, direct ophthalmoscopy and measurement of visual evoked potentials (vep). the tenets of the declaration of helsinki were followed, institutional ethical committee approval was granted, patients were informed about the doppler method ; and an informed consent form was signed by each participant. on was diagnosed by an expert ophthalmologist (ad). cases of acute unilateral on were identified according to the degree of decrease in visual acuity, impaired perimetry findings, slight swelling of the optic disc and facultative retro- or parabulbar pain, afferent papillary defect and delayed vep responses. the contralateral eye in all patients was un - affected by clinical signs of on before the study began. however, we could not exclude the possibility that subclinical damage to the contralateral optic nerve may have occurred, although the veps in these eyes were normal. multiple sclerosis (ms) was presumed to be the cause of on in 18 patients, with no obvious cause in the remaining five. the best corrected visual acuity of the affected eyes varied from counting fingers to 20 / 20 ; the best corrected visual acuity of unaffected eyes was at least 20 / 20 in all cases. the systolic and diastolic blood pressures measured during blood flow velocity investigations did not exceed 130 and 85 mmhg, respectively. orbital doppler and gray - scale sonography was performed within one to seven days of presentation, prior to the initiation of corticosteroids treatment. cdi of the eye was performed in all individuals by two expert sonographers (mk and mr) using a colour doppler unit and a 7.5- to 10-mhz linear - array transducer (model g-60 ; siemens, erlangen, germany). the nerve diameter was measured on both sides, with the unaffected nerve serving as a control. a difference in nerve diameter of 0.3 mm or more, compared with the contralateral side, was defined as a sign of nerve thickening. the patients were examined in the supine position in order to avoid any pressure on the eye. sterile coupling gel was applied to their closed eyelids, with the examiner 's hand resting on the orbital margin to minimize pressure on the globe, and real - time gray - scale and colour - flow images were obtained. we obtained peak systolic velocity (psv) and end - diastolic velocity (edv) measurements in the oa, cra and pcas of both orbits and used these to calculate the vascular resistance (expressed by the ri and pi) using the formulas ri = (psv - edv) / psv and pi = (psv - edv / vmean, where vmean = 1/3 (psv - edv) + edv, in all patients. signals from short pca can be located in the lateral and medial sections of the eyeball and since numerous branches of short pcas were available for assessment, only the medial branch was chosen for analysis in this study. on the basis of an estimated standard deviation of 1.5 and accounting for pair - wise comparisons, we determined that 23 patients would be required to yield 80% power to detect (with a two - sided alpha of 0.05) a 1.0 mm mean difference in optic nerve diameter. psv, edv, ri, and pi obtained from the oa, cra, pcas, and the optic nerve diameter in the orbit with on were compared with those from the contralateral orbit without on using a paired student 's t - test. the utility of optic nerve diameter, psv, edv, ri, and pi in diagnosing on was examined by analysis of the area under receiver operating characteristic (roc) curves, with sensitivity was plotted as a function of 1- specificity. areas under the roc curves were compared using the algorithm developed by delong.. all tests for statistical significance were two - tailed and performed assuming a type i error probability < 0.05. twenty - three consecutive, previously untreated patients with acute unilateral on were recruited from the ophthalmology outpatient clinics of isfahan university of medical sciences, iran between october 2009 and february 2010. these included four men and 19 women, with a mean (standard deviation [sd ]) age of 27.2 (7.0) years, ranging from 19 to 47 years. the inclusion criterion was typical clinical presentation of acute unilateral on before the age of 50 years and exclusion criteria were bilateral on, recurrent on and any disease or anomaly of the contralateral eye. no patient had a history of any major systemic diseases, including cardiovascular disease, arterial hypertension, hyperlipidemia or diabetes mellitus. all patients underwent neurologic and ophthalmologic examinations including visual acuity assessment, direct ophthalmoscopy and measurement of visual evoked potentials (vep). the tenets of the declaration of helsinki were followed, institutional ethical committee approval was granted, patients were informed about the doppler method ; and an informed consent form was signed by each participant. cases of acute unilateral on were identified according to the degree of decrease in visual acuity, impaired perimetry findings, slight swelling of the optic disc and facultative retro- or parabulbar pain, afferent papillary defect and delayed vep responses. the contralateral eye in all patients was un - affected by clinical signs of on before the study began. however, we could not exclude the possibility that subclinical damage to the contralateral optic nerve may have occurred, although the veps in these eyes were normal. multiple sclerosis (ms) was presumed to be the cause of on in 18 patients, with no obvious cause in the remaining five. the best corrected visual acuity of the affected eyes varied from counting fingers to 20 / 20 ; the best corrected visual acuity of unaffected eyes was at least 20 / 20 in all cases. the systolic and diastolic blood pressures measured during blood flow velocity investigations did not exceed 130 and 85 mmhg, respectively. orbital doppler and gray - scale sonography was performed within one to seven days of presentation, prior to the initiation of corticosteroids treatment. cdi of the eye was performed in all individuals by two expert sonographers (mk and mr) using a colour doppler unit and a 7.5- to 10-mhz linear - array transducer (model g-60 ; siemens, erlangen, germany). the nerve diameter was measured on both sides, with the unaffected nerve serving as a control. a difference in nerve diameter of 0.3 mm or more, compared with the contralateral side, was defined as a sign of nerve thickening. the patients were examined in the supine position in order to avoid any pressure on the eye. sterile coupling gel was applied to their closed eyelids, with the examiner 's hand resting on the orbital margin to minimize pressure on the globe, and real - time gray - scale and colour - flow images were obtained. we obtained peak systolic velocity (psv) and end - diastolic velocity (edv) measurements in the oa, cra and pcas of both orbits and used these to calculate the vascular resistance (expressed by the ri and pi) using the formulas ri = (psv - edv) / psv and pi = (psv - edv / vmean, where vmean = 1/3 (psv - edv) + edv, in all patients. signals from short pca can be located in the lateral and medial sections of the eyeball and since numerous branches of short pcas were available for assessment, only the medial branch was chosen for analysis in this study. on the basis of an estimated standard deviation of 1.5 and accounting for pair - wise comparisons, we determined that 23 patients would be required to yield 80% power to detect (with a two - sided alpha of 0.05) a 1.0 mm mean difference in optic nerve diameter. psv, edv, ri, and pi obtained from the oa, cra, pcas, and the optic nerve diameter in the orbit with on were compared with those from the contralateral orbit without on using a paired student 's t - test. the utility of optic nerve diameter, psv, edv, ri, and pi in diagnosing on was examined by analysis of the area under receiver operating characteristic (roc) curves, with sensitivity was plotted as a function of 1- specificity. areas under the roc curves were compared using the algorithm developed by delong.. all tests for statistical significance were two - tailed and performed assuming a type i error probability < 0.05. on involved the right eye in 12 patients (52.2%) and the left eye in the remaining 11 (47.8%) patients. of the 23 eyes with on, 20 (86.9%) showed a thickening of the affected optic nerve. only three eyes with on had a difference in nerve diameter less than 0.3 mm. the increase in diameter of the affected nerve compared with the unaffected contralateral nerve averaged (sd) 1.1 (0.6) mm (range, 0.1 to 2.2 mm). when the cut - off value for nerve pathology was set at a nerve diameter of 4 mm, 15 (65.2%) of the affected nerves and 100% of the unaffected nerves were correctly assessed. the mean (sd) values of parameters obtained from cdi measurements in the oa, cra, pcas and optic nerve in the 23 pairs of eyes with and without on are shown in table 1. as expected, the mean (sd) optic nerve diameter was 4.1 (0.8) mm for eyes with on and 3.0 (0.4) mm for eyes without on (p < 0.001). there were no statistically significant differences between eyes with and without on in terms of psv, edv, ri, and pi in oa and cra measurements. the mean ri in the pcas was slightly lower in eyes with on than in control eyes (0.60 vs. 0.64, p < 0.05). the areas under the roc curves for the occurrence of on for orbital blood flow velocities and resistance indices of oa, cra, pcas, and optic nerve diameter are shown in fig. 1 and table 2. the area under the roc curve was 0.928 (95% ci [confidence interval ] : 0.843, 0.101) for optic nerve diameter. the areas under the roc curves were 0.497 (95% ci : 0.306, 0.688) for psv, 0.504 (95% ci : 0.314, 0.695) for edv, 0.534 (95% ci : 0.345, 0.722) for ri and 0.503 (95% ci : 0.311, 0.695) for pi in oa. psv, edv, ri, and pi of the oa, cra, and pcas covered a similar area. the edv of the cra and pcas had areas slightly but not significantly larger than that of the other blood flow parameters. in this study we found no significant difference in psv, edv, or pi of the oa, cra, and pcas between eyes with and without on. the mean ri in the pca was slightly lower in the eyes with on than in the contralateral eyes. thickening of the optic nerve results from the inflammation in on and has been described previously. the observed thickening is likely to be a major cause of the initial loss of visual acuity. the enlarged optic nerve in on compresses the oa within the optic canal and this compression may contribute to the impairment of orbital hemodynamics. in this study the ri is angle - independent and provides a good measurement by which to quantify vascular resistance in circulation, particularly in tortuous vessels like the pca. few studies have performed cdi to assess orbital blood flow velocities and optic nerve diameter in on ; these were usually of limited sample size and the results produced were inconsistent. the inconsistency may be explained in part by differences in patient characteristics, the cause of on, definitions of on, blood flow velocity measurements and the amount of time elapsed between the acute attack and examination. while differences in hemodynamic parameters over the course of the disease may partly account for the range of results seen in the literature, it is much more likely that the main component of variance in on doppler studies results from differences in instrumentation and technique. large foot - print transducers, in the 7.5- to 10.0-mhz range, with poor lateral resolution, placed directly on the closed eyelid at the orbital ridge while not specifically avoiding the lens will not yield optimal measurements. evaluated only the resistive indices of the cra ; they found resistive indices increased in the affected side and attributed the difference to nerve swelling and the resultant resistance to flow. our findings also differ from those of karaali., who found that psv, edv, and ri in the oa are increased in patients with acute on although the velocities and resistive indices of the cra in affected and normal eyes did not differ significantly. also reported that psv, pi, and ri in the oa are increased in patients with acute on although the edv in affected and normal eyes did not differ significantly. furthermore, pache. reported a significant reduction in the psv and edv of the oa, pcas, and cra in patients with ms compared with healthy controls. modrzejewska. also reported statistically significant diminishing blood flow velocity parameters in the eyeball arteries of patients with both ms and on. they did not observe any changes in vascular resistance indices when compared with the control group. the absence of abnormalities in vascular resistance could be due to a comparatively long passage of time between the clinical manifestation of on and the study. reported that in patients with ms, the mean psv, edv, and ri in the oa of eyes with on were not significantly different from those in unaffected fellow eyes and healthy control eyes. the mean edv in the cra was lower and the mean resistivity indices in the cra and pca were higher in eyes with on than in control eyes. they found that orbital hemodynamics in all retrobulbar vessels did not significantly differ from normal controls in patients with compressive, inflammatory, toxic or hereditary optic neuropathy. our findings indicate that optic nerve diameter is the most reliable and practical predictor of on. the pathophysiology of on has not been fully described, but thickening of the nerve in association with demyelination and inflammation may compress the blood vessels within the optic canal. alternatively, vasospasm due to an increased plasma level of endothelin-1, a potent vasoconstrictor, may result in vasospasm and vascular dysregulation. this could cause an increase in resistance to flow in the artery and through ischemia lead to eventual exoplasmic stasis and visual loss. evaluation with gray - scale sonography of the optic nerve and cdi of orbital hemodynamics in patients with acute on, combined with proof of decreased ri in pcas may facilitate an accurate diagnosis. one limitation of this study is that blood flow measurements were only performed in the contralateral eyes of the patients without an additional control group. however, as values of orbital blood flow velocity are variables within the healthy population, any differences in values between the orbit affected by on and the unaffected orbit are highly significant. use of the contralateral eye as an internal control therefore seems practical since cdi measurement is known to be influenced by cardiac output and the patient 's stress level. moreover, on swelling may influence cdi readings to an unknown degree independent of changes in blood flow due to the altered scattering properties of tissue. additionally, akarsu. demonstrated that there was a similarity in blood flow velocities between contralateral unaffected eyes and healthy control eyes. the patients in our series were under 50 years old, an age at which glaucoma is uncommon and intraocular pressure was normal in all patients. an experimental study by guthoff. showed that an increase in intraorbital pressure leads to a decrease in cra flow. great care was thus taken to apply as little pressure as possible to the patient 's eye during cdi examination. even though the study included the thorough examination of 23 pairs of eyes, the findings should be interpreted with caution due to the relatively small sample size. although we have not carried out any special reliability studies, cdi measurements of the eye vessels and between the right and left eyes have been shown to be reproducible over time. in conclusion, our findings indicates that gray - scale sonography is a non - invasive and repeatable diagnostic method, which reveals significant optic nerve thickening on the side affected by on and could play a role in disease diagnosis. no unilateral differences were observed in the average psv, edv, ri, or pi of the oa and cra. the reduced ri in pcas may indicate disturbances in retinal and choroidal circulation in patients with on. further studies with larger groups of patients are needed with age- and sex - matched control groups in order to better understand the role of retrobulbar hemodynamics in the pathogenesis of on.	purposeto evaluate orbital blood flow velocities and optic nerve diameter with doppler and gray - scale sonography in patients with acute unilateral optic neuritis (on).methodsorbital doppler and gray - scale sonography was performed in 46 eyes of 23 patients aged 19- to 47-years with acute unilateral on. on was diagnosed by an ophthalmologist on the basis of clinical presentation, presence of decreased visual acuity and assessment of visual evoked potentials. the peak systolic velocity (psv) and end - diastolic velocity (edv), as well as the resistance index (ri) and pulsatile index (pi) of the ophthalmic artery (oa), central retinal artery (cra), posterior ciliary arteries (pcas) and optic nerve diameter were measured in both eyes. we compared results from affected and unaffected eyes using the paired t - test. the area under the receiver operating characteristic (roc) curves was used to assess the diagnosis of on based on measured blood flow parameters of the oa, cra and pcas and optic nerve diameter.resultsthe mean (standard deviation) optic nerve diameter in eyes with on was 4.1 (0.8) mm, which was significantly larger than the 3.0 (0.4) mm diameter measured in unaffected control eyes (p 0.05). the mean ri in the pcas was slightly lower in the eyes with on than in the contralateral eyes (0.60 vs. 0.64, p < 0.05). the area under the roc curves indicated that optic nerve diameter was the best parameter for the diagnosis of on.conclusionsoptic nerve diameter was related to on, but orbital blood flow parameters were not.
hereditary tyrosinemia type i (hti) is an autosomal recessive metabolic disorder due to deficiency of fumarylacetoacetate hydrolase enzyme. the diagnosis is based on high blood tyrosine level and abnormal urinary excretion of succinylacetone (sa) (1). according to onset of symptoms ht1 is classified in acute (6 months) forms (2). most common causes of death are liver failure and bleeding, hepatocellular carcinoma (hcc) and pseudo porphyric crisis with respiratory failure (3). since 1992, the 2- (2-nitro-4trifluoro - methyl benzoyl)-1, 3-cyclohexanedione (ntbc) has offered as a new promising tool for the treatment (4, 5)., there are few reports of the long - term prognosis (5, 7). this study reports beside its clinical and biochemical presentation, the outcome of ntbc [2- (2-nitro-4-trifloro - methylbenzoyl)-1, 3-cyclohexanedion ] treatment of the disease and evaluates its biochemical markers in 16 pediatric libyan patients. a retrospective clinical study was undertaken on 16 libyan patients with hereditary tyrosinemia type i (hti) who were diagnosed or referred to a single center at elkhadra hospital, one of the main referral hospitals in western libya, which provides medical services to pediatric age groups up to 16 years old. our orientation was based on the presence of rickets with hepatomegaly or liver disease with high alpha fetoprotein (afp). two patients were detected by screening at the age of one week and 1.5 months because of older affected siblings. the diagnosis of hti was confirmed by high tyrosine level in blood and sa urinary excretion by liquid chromatography tandem mass spectrometry (lc - ms / ms). the investigation (blood for amino acids, urine for organic acids and sa or dried blood spots for the same tests) were sent to laboratories and were done either in tunisia, germany, france or lebanon. x - ray of bones, echocardiography, abdominal ultrasonography and when indicated, abdominal ct scan were monitored. follow up visits were done iat least monthly in the first 6 months, then every 3 months thereafter. one patient was treated with diet protein restriction due to unavailability of ntbc on diagnosis ; and 15 patients were treated with ntbc (initially 1 mg / kg / day in 2 divided doses, the dose thereafter was adjusted according to clinical response ranging 0.5 - 2 mg / kg / day) (8, 9). prescribed diet consisted of avoiding high protein, continuing breast feeding and employing a special milk formula. the goal of the diet was to maintain the blood levels of tyrosine below 500 mol / l (by restricted tyrosine and phenylalanine each one dose not exceed 200 mg / day). supplementation by fat soluble vitamins d and k in liver dysfunction and phosphate in hypophosphatemia were provided. with spss program version 16 the paired samples t - test was used, p value < 0.05 was considered statistically significant. with spss program version 16 the paired samples t - test was used, p value < 0.05 was considered statistically significant. sixteen patients were diagnosed with hti, rate of diagnosis was 1 - 2 children / year, 81.2% (n = 13) were born to consanguineously married couples, and 2 families had 2 affected children each. the median age at onset of first symptoms was 4.5 months (range 1 week - 8 months). median age of first visit presentation to our unit was 7 months (range : 1 week to 38 months). the median age at diagnosis was 8 months (range 1 week - 40 months). the median age at starting treatment was 9 months (range 1 week - 43 months), the median interval between diagnosis and treatment was 0.5 months (range 0 - 11 months). the main clinical presentation at first visit was hepatomegaly, jaundice, rickets with prolonged pt and hypophosphatemia which were seen in 14 (87.5%) patients. bleeding tendency, ascites and edema with abnormal nodular liver imaging were seen in 3 (18.7%) patients with liver failure. anemia and thrombocytopenia (5 patients) were transient and normalized after initiation of treatment. other associated less common presentations were night irritability (3 patients), severe failure to thrive (3 patients), multiple fractures (1 patient) (figure 1). no child had symptoms related to cardiovascular or central nervous system (tables 1 and 2). at presentation, extremely high afp (figure 2) and high alp were seen in all patients, the ggt was high in 87.5% (n = 14) but was normal for age in 2 patients who were diagnosed by screening, alt was high in 68.7% (n = 11) and ast in 56.2% (n = 9) (table 2). overall 9-year survival rate has been 81.2% whereas it was 86.6% in ntbc treated patients. abbreviations : alp, alkaline phosphatase ; alt, alanine aminotransferase ; ast, aspartate aminotransferase ; ggt, gamma glutamyl transferase. we compared the onset of treatment and the drop of afp levels in two groups. the first group was five patients with poor response who began treatment at median of 13 months post onset of first symptoms (range : sex to 35 months). the second group was 11 patients with good response who began treatment at median of three months post onset of first symptoms (range : one to 14 months). we observed that by the third month post treatment that level of afp in the second group had faller by more than 50% of pre - treatment values in 90% (n.10) of patients. we observed this drop in only 20% (n.1) of the first group (p = 0.003). the outcome of ntbc treatment after one year in 13 survived patients was as follows : pt was the first liver function marker that showed normalization (median 14 days, range 0 - 240 days), while other biochemical liver markers such as transaminases (gpt, got), ggt, alp, bilirubin, serum albumin, afp and renal function took longer to improve. in subacute form, normalization of afp was 50% in the first year and 100% in the second year, pt was normalized 100% and other liver markers 75% ; one patient had abnormal liver imaging (hepatomegaly without nodules which sustained for longer). in chronic form, normalization of afp was 16.6% and 83.3% in the 3rd year and later, the pt was 83.3% and other liver function markers were normalized in 66.6% ; persistence of abnormal liver imaging (hepatomegaly) was seen in one patient. in none of forms increase of afp was seen (figure 2). the constant features were decreased rates of weight gain and poor dentition, caries or delayed eruption of deciduous teeth occurred in all patients, the first permanent teeth erupted at due time and were healthy. neuropsychosocial development, motor function and speech were normal for age in all survived patients, but are not assessed by any standardized test system. three patients who presented severe liver cirrhosis (splenomagly, esophageal varices with pt 21% to 24%) and persisting high afp died. they were at onset of symptoms 1, 5 and 8 months, and at diagnosis 6, 6, 40 months old. the treatment was started at 7 months (diet only), 8 months and 43 months (ntbc and diet). the interest for genetic disorders is relatively recent in libya, where many sick babies or unexplained infant deaths may be attributable to inborn errors of metabolism. the preliminary investigations fail to give a diagnostic clue, the patients actually pass away without metabolic workup because lack of awareness and limited diagnostic facilities. hti is a genetic autosomal recessive disorder ; the risk is greater in mating relationships where the parents are close relatives. libya displays some of the highest consanguinity rates in the world. according to 2008 estimate it was as high as 48.4% in a total population of 6.18 million (11). considering that 16 patients all of whom, were referred to a single hospital, with 81.2% consanguinity rate, it seems that hti is one of the most commonly recognized rare disorders in libya. from other countries in north africa, namely tunisia, more than 70 cases were reported since 1987 with 25 ntbc treated patients since 2000 (dr. h azzouz, tunisia experience in management of hti, memg 2012), from algeria 30 treated patients and morocco 3 patients (swedish orphan communication). the first option is dietary treatment that results in a poor outcome and caries a high risk of hcc, the second option is the liver transplantation which became the efficient treatment with good survey, and the third option is the ntbc which has been available since 1992, has transformed the prognosis and has obviously improved the survival rate of hti in all forms particularly in acute liver failure. ntbc has been used since 2001 as treatment for hti in libya. of patients exposed to ntbc, 60% of patients (n = 9) with more than 3 years, 40% (n = 6) with more than 6 years and 2 patients with more than 8 years of ntbc exposure. in none of the patients liver transplant three patients died, the cause of death was either late diagnosis or delayed starting of treatment but hcc was not ruled out (12, 13). we found that elevated ggt was a better marker (figure 3) with respect to following : 1) at presentation it was high and increased earlier than other enzymes in 87.5% of patients. 2) remained longer high in the first 6 months after starting treatment except in 2 asymptomatic screened babies. 4) it was the first enzyme that raised alarm when treatment in patients who previously had responded well was inadequate. all patients showed remarkable improvement of the signs of rickets after one year of treatment. the renal tubulopathy associated with hti treated with ntbc is usually reversible (14). no patient presented with neurological manifestation but we faced porphyric like crisis in one very well responding, early treated asymptomatic screened patient after 6 years of ntbc treatment there occurred a sudden acute shortage in ntbc, his -aminolevulinic acid was high, more than 20 times above the upper limit of normal, the recovery was achieved with reintroducing ntbc and supportive treatment. the side effects of ntbc, were seen in most of our patients leucopenia and thrombocytopenia, were transient but we never observed toxic signs of the eyes such as keratitis, photophobia and corneal opacities (15), nor did we observe skin exfoliative dermatitis a sign of high blood tyrosine associated with uncontrolled diet restriction. 7. introducing of neonatal metabolic screening, possibility of liver transplantation, promotion of local metabolic laboratories and a molecular testing is needed.	background : hereditary tyrosinemia type i (hti) is a metabolic disease caused by deficiency of fumarylacetoacetate hydrolase enzyme.objectives:this study reports beside its clinical and biochemical presentation, the outcome of ntbc [2- (2-nitro-4-trifloro - methylbenzoyl)-1, 3-cyclohexanedion ] treatment of the disease and evaluates its biochemical markers in 16 pediatric libyan patients.patients and methods : the diagnosis was based on presence of high tyrosine levels in blood and succinylacetone in urine.results:the consanguinity rate was 81.2%, the median age at onset, at diagnosis and at starting treatment were 4.5, 8, and 9.5 months respectively. at presentation hepatomegaly, jaundice, rickets and high gamma glutamyl transferase (ggt) were observed in 87.5% of patients. all patients had extremely high alpha fetoprotein (afp) and high alkaline phosphatase (alp) levels. fifteen patients were treated with ntbc, normalization of pt (prothrombine time) was achieved in average in 14 days. the other biochemical parameters of liver function (transaminases, ggt, alp, bilirubin and albumin) took longer to improve and several months to be normalized. survival rate with ntbc was 86.6%. patients who started treatment in a median of 3 months post onset observed a fast drop of afp in 90.6% of patients (p = 0.003). abnormal liver function and rickets were the common presentations, ggt was an early cholestatic sensitive test. alp was constantly high even in asymptomatic patients.conclusions:in ht1 a faster dropping of afp is a marker of good prognosis.
prostate cancer is the most prevalent cancer and the third most common cause of mortality in men in western countries. transrectal ultrasonography - guided prostate biopsy (trus - bx) is an essential procedure for diagnosing prostate cancer but may cause variable complications, including pain, hematuria, hemospermia, urethral injury, and urinary tract infection. among the complications, infective complications are clinically important because these can cause prostatitis or urosepsis, which are sometimes fatal to patients. the incidence rate of infective complications after trus - bx was reported to range from 0.1% to 7% and has increased during the past 10 years. for example, the american urological association (aua) guideline recommends antibiotic prophylaxis with fluoroquinolone for less than 24 hours as the first - line therapy and aminoglycoside combined with metronidazole or clindamycin for less than 24 hours as the second - line therapy. the european association of urology (eau) guideline also recommends oral or intravenous antibiotic prophylaxis before biopsy, and quinolone is recommended as the drug of choice. however, these recommendations would not be appropriate in a region where the prevalence of quinolone - resistant escherichia coli is high. the recent increase in infective complications is possibly due to an increase in quinolone - resistant e. coli prevalence in the community. because overuse of antibiotics has not been strictly controlled in korea, a recent epidemiology study reported that the resistance rate to ciprofloxacin was over 24% in cystitis patients. for this reason, korean urologists hardly follow the aua or eau guideline and instead use various combinations of antibiotics to prevent infective complications. considering the increase in quinolone - resistant pathogens worldwide, it is clinically significant to know the pattern of antibiotic prophylaxis in korea, where the prevalence of quinolone - resistant e. coli is already high. such data would give insight into the future of antibiotic prophylaxis in other countries where the quinolone resistance rate is still low but increasing. therefore, we investigated the real practice of antibiotic prophylaxis to prevent infective complications of trus - bx in korea. we sent an e - mail to the urology departments of 77 hospitals in korea. these hospitals were secondary or tertiary referral hospitals and covered most prostate biopsy cases in korea. the contents of the e - mail included questions about the real practice of trus - bx as follows : (1) which department has performed trus - bx ? (department of radiology or urology), (2) how has biopsy been performed ? (inpatient or outpatient setting), (3) which type of enema has been performed ? (4) has betadine enema been performed ? (5) which kinds of antibiotics have been used prior to biopsy ? (6) which kinds of antibiotics have been used after the procedure ? (7) how long did the patients take the medicine after prostate biopsy ? we analyzed the contents of the returned e - mails. according to the responses received, the prophylactic drugs in practice before and after trus - bx were classified according to the type of antibiotic (quinolone, cephalosporin, aminoglycoside, and metronidazole), and we investigated the prevalence of each antibiotic regimen. in addition, duration of postbiopsy medication and the identity of the antibiotics used before and after biopsy were evaluated. all statistical analyses were performed by using spss ver. 17.0 (spss inc., chicago, il, usa), and the prevalence was presented as the number of hospitals with percentages. a total of 54 hospitals replied to our e - mail ; the response rate was 70.1%. two thirds of the responding hospitals indicated that the department of urology performed the prostate biopsy procedures (37 hospitals, 68.5%), and patients were admitted for trus - bx in 33 hospitals (61.1% ; fig., various types of enema or suppositories were used in 48 hospitals (88.8%), and disinfection with povidone - iodine was used in 29 hospitals (53.7% ; fig. 2). various types of prophylactic antibiotics prior to prostate biopsy were used in each hospital (table 1). a quinolone alone regimen was the most common (26 hospitals, 48.1%), followed by cephalosporin alone (11 hospitals, 20.4%), and aminoglycoside alone (5 hospitals, 9.3%). a combination of two or more antibiotics was used in 12 hospitals (22.2%). the doctors of all hospitals that participated in this study prescribed oral antibiotics after trus - bx (table 2). the most common type of postbiopsy medication was quinolone alone (42 hospitals, 77.8%). a cephalosporin alone regimen was used in 11 hospitals (20.4%), and only 1 hospital answered that oral quinolone and cephalosporin were routinely prescribed simultaneously. the treatment duration of postbiopsy antibiotics also varied among the hospitals (table 2). routine protocols of most hospital (43 hospitals, 79.6%) were treatment for 4 or more days with oral antibiotics after trus - bx. among the 26 hospitals that chose quinolone alone before biopsy, 1 hospital (3.8%) changed the type of antibiotic to cephalosporin after biopsy. the other hospitals maintained an identical type of antibiotic during the post - biopsy period. among the 11 hospitals using cephalosporin alone prior to trus - bx in contrast, the other 8 hospitals adhered to the same type of antibiotic used for the initial prophylaxis. postbiopsy quinolone was used in all 3 hospitals that administrated aminoglycoside alone before trus - bx. overall, the proportion of hospitals using prophylaxis with a single type of antimicrobial agent before and after prostate biopsy was 61.1% (33 hospitals). on the other hand, various types of prophylactic antibiotics prior to prostate biopsy were used in each hospital (table 1). a quinolone alone regimen was the most common (26 hospitals, 48.1%), followed by cephalosporin alone (11 hospitals, 20.4%), and aminoglycoside alone (5 hospitals, 9.3%). a combination of two or more antibiotics was used in 12 hospitals (22.2%). the doctors of all hospitals that participated in this study prescribed oral antibiotics after trus - bx (table 2). the most common type of postbiopsy medication was quinolone alone (42 hospitals, 77.8%). a cephalosporin alone regimen was used in 11 hospitals (20.4%), and only 1 hospital answered that oral quinolone and cephalosporin were routinely prescribed simultaneously. the treatment duration of postbiopsy antibiotics also varied among the hospitals (table 2). routine protocols of most hospital (43 hospitals, 79.6%) were treatment for 4 or more days with oral antibiotics after trus - bx. among the 26 hospitals that chose quinolone alone before biopsy, 1 hospital (3.8%) changed the type of antibiotic to cephalosporin after biopsy. the other hospitals maintained an identical type of antibiotic during the post - biopsy period. among the 11 hospitals using cephalosporin alone prior to trus - bx in contrast, the other 8 hospitals adhered to the same type of antibiotic used for the initial prophylaxis. postbiopsy quinolone was used in all 3 hospitals that administrated aminoglycoside alone before trus - bx. overall, the proportion of hospitals using prophylaxis with a single type of antimicrobial agent before and after prostate biopsy was 61.1% (33 hospitals). on the other hand, various combinations of drugs were used in 21 hospitals (38.9%). the incidence of acute prostatitis related to trus - bx has been reported to range from 0.1% to 7%. in korea, the incidence of biopsy - related prostatitis was reported to range from 1.4% to 1.9%. various studies have been undertaken to reduce the incidence of acute prostatitis after trus - bx. one study reported that rectal preparation prior to biopsy with bisacodyl decreased biopsy - induced infectious complications significantly compared with a non - rectal - preparation group. an intrarectal mixture of povidone - iodine and lidocaine gel also significantly decreased infectious complications in another study. recently, issa. described that formalin disinfection of a biopsy needle after each core reduces the incidence of urinary tract infection and sepsis. among these prophylactic methods, antibiotic prophylaxis has been the standard method of preventing infectious complications. in particular, fluoroquinolones have traditionally been used as the primary prophylactic agent owing to their excellent prostatic penetration. in the past, these agents also provided good coverage against the key pathogens implicated in infections after prostate biopsy. analyzed a total of 1,994 strains from patients with community - acquired urinary tract infection from 34 hospitals in korea from january 2008 to june 2009. in that study, the resistance rate to ciprofloxacin was reported to range from 20% to 38% in community - acquired uncomplicated cystitis and showed a tendency to increase. the overall resistance rate to ciprofloxacin rapidly increased from 15.2% in 2002 to 24.8% in 2009. furthermore, ciprofloxacin - resistant e. coli has been shown to be more prevalent in complicated urinary tract infection (40.9%) than in uncomplicated urinary tract infection (24.8%) in korea. recent studies have reported that the prevalence of quinolone - resistant e. coli has also increased in western countries. however, the rate of quinolone resistance is lower than in korea. in the united states, fluoroquinolone - resistant bacteria were identified in 22% of samples from rectal swab cultures before trus - bx. in that study, asian men had a higher risk of resistant rectal flora colonization (odds ratio, 2.8). in american men who developed acute prostatitis after trus - bx, the fluoroquinolone - resistant rate was reported to be 57.1%. in korea, a study reported that 2.0% of patients who underwent trus - bx developed acute prostatitis, and nearly all culture - positive specimens (96.3%) were identified as ciprofloxacin - resistant pathogens. in that study, most quinolone - resistant pathogens were sensitive to cephalosporin and aminoglycoside. therefore, several studies have evaluated the effect of cephalosporin combined with quinolone on the prevention of infectious complications after trus - bx. some reports have suggested that adding aminoglycoside is helpful for preventing infectious complications in areas with a high antibiotic resistance rate. the aua guideline does not recommend using multiple antibiotics during prostate biopsy. in our study, 21 hospitals (38.9%) used two or more antimicrobial agents during trus - bx. cephalosporin alone or combined with other antibiotics was used in 19 hospitals (35.2%), and aminoglycoside alone or combined with other antibiotics was used in 13 hospitals (24.1%). only 25 hospitals (46.2%) followed the aua guideline recommending fluoroquinolone alone before and after trus - bx. no hospitals used alternative antimicrobials (aminoglycoside with metronidazole or clindamycin). regarding the duration of antibiotic treatment, fewer hospitals adhered to aua guidelines. prophylactic antibiotic use of less than 24 hours was used by only 7.4% of the hospitals in our study, although several studies have suggested that there are no significant benefits of long - term use. a recent cochrane review revealed that there was no clinically nor statistically significant difference between a short course or a single - dose regimen compared with a longer course. our study revealed that a quinolone only regimen was the single most common method for prophylaxis, but less than half of hospitals used a quinolone only regimen for prophylaxis. these results showed that most hospitals did not follow the aua or eau guidelines for various reasons. first, we could not evaluate the incidence of infectious complications nor the changes in complications after modifying the antibiotic regimen at each hospital because this study was a cross - sectional observational study. furthermore, we could not assess why the current practice was selected in each hospital. because there are no randomized controlled trials comparing the effectiveness of antibiotics for trus - bx in korean populations, and because current recommendations for antibiotic prophylaxis for trus - bx are derived from studies performed in western countries, we can assume that the current prophylactic antibiotic regimens are based on clinical experience. therefore, the results of this study do not mean that other antibiotic regimens are more effective for preventing acute prostatitis after trus - bx than the quinolone - alone regimen. nonetheless, this is still the first study that has reported the practical use of antibiotics during trus - bx. as such, the results of this study could be helpful for establishing a health policy in countries with increasing antibiotic resistance. the aua recommendation was not followed by most hospitals and a combination regimen was used by nearly half of the responding hospitals in korea. this clinical practice pattern might be a result of the struggle to reduce infective complications after trus - bx in a country where the quinolone resistance rate is high. to justify this empirical use of antibiotics,	purposetransrectal ultrasonography - guided prostate biopsy (trus - bx) is an essential procedure for diagnosing prostate cancer. the american urological association (aua) guideline recommends fluoroquinolone alone for 1 day during trus - bx. however, this recommendation may not be appropriate in regions where the prevalence of quinolone - resistant escherichia coli is high. we investigated the real practice of antibiotic prophylaxis for trus - bx in korea.materials and methodsa total of 77 hospitals performing trus - bx were identified and an e - mail was sent to the urology department of those hospitals. the questions in the e - mail included the choice of antibiotics before and after the procedure and the duration of antibiotic therapy after trus-bx.resultsa total of 54 hospitals (70.0%) responded to the e - mail. before trus - bx, all hospitals administered intravenous antibiotic prophylaxis. the percentage of hospitals that used quinolone, cephalosporin, and aminoglycoside alone was 48.1%, 20.4%, and 9.3%, respectively. the percentage of hospitals that used two or more antibiotics was 22.2%. after biopsy, all 54 hospitals prescribed oral antibiotics. the percentage of hospitals that prescribed quinolone alone, cephalosporin alone, or a combination of two or more antibiotics was 77.8%, 20.4%, and 1.8%, respectively. the duration of antibiotic use was more than 3 days in most hospitals (79.6%). only four hospitals (7.4%) followed the aua recommendation of a 1-day regimen.conclusionsthe aua recommendation was not followed by most hospitals in korea. this clinical behavior might reflect the high quinolone resistance rate in korea, and further studies on the most efficient prophylactic antibiotics after trus - bx in korea are warranted.
preservation of the remaining vital portion of curiously exposed pulpal tissue in primary teeth, where the demand is to keep a functioning tooth in site, was one of the most frequent problems in pediatric dentistry.1 to solve this problem pulpotomy therapy was introduced, developed and classified according to treatment objectives.2 pulpotomy involves amputation of the coronal portion of affected or infected dental pulp, treatment of the remaining vital radicular pulp tissue surface should preserve the vitality and function of all or part of the remaining radicular portion of the pulp.3 furthermore, it is an accepted procedure for treating both primary and permanent teeth with carious pulp exposures, several materials have been using for capping the radicular pulp after pulpotomy, these included formocresol, glutaraldehyde,4 ferric sulfate,5 the collagen material,6 and mineral trioxide aggregate.7 however, none of them had met the same degree of effectiveness and success rate as formocresol, possible hazards of formocresol (cytotoxicity, carcinogenicity, immunologic, biochemical, mutagenic, and teratogenic changes) in the host have been reported.8 moreover, it produced enamel defects in the permanent successors.9 in this regard, the demand for natural medicament to replace formocresol as a pulp dressing material became imperative. the antibacterial activity of a. sativum is mediated by allicin through enzymatic activity of allinase (a cysteine sulfoxidelyase). a wide range of therapeutic effects reported for garlic is mainly from allicin and other thiosulfinates. the antibacterial effects of fresh garlic extract10 have been thoroughly researched via literature and have been found that the extract will inhibit growth of various gram - positive and gram - negative bacteria.11 it is also reported that the garlic extract has inhibitory potential on isolated multi drug resistant strains of streptococcus mutans from human caries teeth.12 the aim of this study was to compare between the clinical and radiographic effects of a. sativum oil and those of formocresol in vital pulpotomy in primary teeth. the aim of this study was to compare between the clinical and radiographic effects of a. sativum oil and those of formocresol in vital pulpotomy in primary teeth. twenty children age ranged from 4 to 8 years were selected from outpatient clinic of pediatric dentistry department, faculty of dental medicine, al - azhar university, egypt. care giver approval was taken.approval of al - azhar university, faculty of oral and dental medicine, egypt (under number 249/2010). approval of al - azhar university, faculty of oral and dental medicine, egypt (under number 249/2010). a comprehensive history was obtained, and a thorough clinical examination was conducted on each of the selected children. moreover, asking the parents or caregivers about any medical problems that may contraindicate the use of any of the intended procedures. a diagnostic sheet had been made for each child including the personal information, clinical and radiographical evaluations. in every one of those children, a pair of primary molars indicated for pulpotomy patient and parent cooperationabsence of any systemic disease, which would contraindicate pulp therapyexposure of vital pulp after excavation of caries with no clinical evidence of the extensive pulp degeneration or any periapical pathologic conditionabsence of clinical signs or symptoms suggesting a non - vital tooth such as a suppurating sinus, soft tissue swelling, mobility or tenderness to percussionpossibility for establishing a final restoration of the tooth. patient and parent cooperation absence of any systemic disease, which would contraindicate pulp therapy exposure of vital pulp after excavation of caries with no clinical evidence of the extensive pulp degeneration or any periapical pathologic condition absence of clinical signs or symptoms suggesting a non - vital tooth such as a suppurating sinus, soft tissue swelling, mobility or tenderness to percussion possibility for establishing a final restoration of the tooth. molars to be treated were locally anaesthetized using mepecaine - l (a local anesthetic solution containing 20 mg mepivacaine hydrochloride with 0.06 mg levonordefrin hydrochloride). patients were allowed to wait for 10 - 15 min before pulpotomy procedure was done, rubber dam or cotton rolls were used to isolate the designated molar. cavity outline was established with a sterile # 330 high - speed pear - shaped carbide bur with air / water spray. access to a pulp chamber could be detected with a probe, or if the roof of the pulp chamber were sufficiently thin to see the pulpal tissue. when the pulpal exposure was confirmed, the roof of the pulp chamber was removed with a sterile, non - end cutting slow - speed bur. removal of the coronal pulp tissue was achieved with a sterile low - speed carbide round bur and/or sharp, large, spoon excavator. hemostasis was attained by placing small cotton pellet moistened in sterile saline with slight pressure then it was removed. the pulp stumps of molars in the right side were dressed with a cotton pellet damped with a. sativum oil (captin company (cappharm) registration no 952/94 cairo, egypt), and with formocresol (petrpolis - rj - industria brasileira, dentsply, latin america) for molars in the left side for 5 min. then, pulp stumps of molars treated with a. sativum oil were dressed in zinc oxide - a. sativum oil paste, while those of teeth treated with formocresol were dressed with a thick paste prepared by mixing zinc oxide powder with one drop of eugenol.13,14 then, a layer of intermediate restorative material zinc phosphate cement had been placed over the dressing materials. after that, molars were restored with suitable final restoration and then with stainless steel crowns. then, molars were clinically and radiographically evaluated after 6 months, using standard clinical and radiographic criteria. history of pain related to the treated molarssensitivity to percussionteeth mobilitysigns of erythema, swelling, the presence of the fistulous tract in the surrounding gingival tissues and mucosa. history of pain related to the treated molars sensitivity to percussion signs of erythema, swelling, the presence of the fistulous tract in the surrounding gingival tissues and mucosa. periapical radiographs were taken for all treated molars (ultra speed kodak periapical films, size 0 or 1 film). all radiographs taken during the follow - up period were screened for their diagnostic quality prior to being included in radiographic evaluation. acceptable radiographs have non - distorted images of the treated molars and the osseous structures immediately adjacent to the roots. radiographs that did not meet these criteria were excluded from the evaluation. during radiographic evaluation, the following were determined : presence or absence (or reduce in size) of furcation or periapical radiolucencypresence or absence of pathologic internal or external root resorptionpresence or absence of widening in the periodontal membrane. presence or absence (or reduce in size) of furcation or periapical radiolucency presence or absence of pathologic internal or external root resorption presence or absence of widening in the periodontal membrane. statistical analysis was performed between the clinical and radiographic effects of a. sativum oil and those of formocresol in vital pulpotomy in primary teeth using chi - square, and the level of significance was taken at = 0.05. patient and parent cooperationabsence of any systemic disease, which would contraindicate pulp therapyexposure of vital pulp after excavation of caries with no clinical evidence of the extensive pulp degeneration or any periapical pathologic conditionabsence of clinical signs or symptoms suggesting a non - vital tooth such as a suppurating sinus, soft tissue swelling, mobility or tenderness to percussionpossibility for establishing a final restoration of the tooth. patient and parent cooperation absence of any systemic disease, which would contraindicate pulp therapy exposure of vital pulp after excavation of caries with no clinical evidence of the extensive pulp degeneration or any periapical pathologic condition absence of clinical signs or symptoms suggesting a non - vital tooth such as a suppurating sinus, soft tissue swelling, mobility or tenderness to percussion possibility for establishing a final restoration of the tooth. molars to be treated were locally anaesthetized using mepecaine - l (a local anesthetic solution containing 20 mg mepivacaine hydrochloride with 0.06 mg levonordefrin hydrochloride). patients were allowed to wait for 10 - 15 min before pulpotomy procedure was done, rubber dam or cotton rolls were used to isolate the designated molar. cavity outline was established with a sterile # 330 high - speed pear - shaped carbide bur with air / water spray. access to a pulp chamber could be detected with a probe, or if the roof of the pulp chamber were sufficiently thin to see the pulpal tissue. when the pulpal exposure was confirmed, the roof of the pulp chamber was removed with a sterile, non - end cutting slow - speed bur. removal of the coronal pulp tissue was achieved with a sterile low - speed carbide round bur and/or sharp, large, spoon excavator. hemostasis was attained by placing small cotton pellet moistened in sterile saline with slight pressure then it was removed. the pulp stumps of molars in the right side were dressed with a cotton pellet damped with a. sativum oil (captin company (cappharm) registration no 952/94 cairo, egypt), and with formocresol (petrpolis - rj - industria brasileira, dentsply, latin america) for molars in the left side for 5 min. then, pulp stumps of molars treated with a. sativum oil were dressed in zinc oxide - a. sativum oil paste, while those of teeth treated with formocresol were dressed with a thick paste prepared by mixing zinc oxide powder with one drop of eugenol.13,14 then, a layer of intermediate restorative material zinc phosphate cement had been placed over the dressing materials. after that, molars were restored with suitable final restoration and then with stainless steel crowns. then, molars were clinically and radiographically evaluated after 6 months, using standard clinical and radiographic criteria. history of pain related to the treated molarssensitivity to percussionteeth mobilitysigns of erythema, swelling, the presence of the fistulous tract in the surrounding gingival tissues and mucosa. history of pain related to the treated molars sensitivity to percussion signs of erythema, swelling, the presence of the fistulous tract in the surrounding gingival tissues and mucosa. periapical radiographs were taken for all treated molars (ultra speed kodak periapical films, size 0 or 1 film). all radiographs taken during the follow - up period were screened for their diagnostic quality prior to being included in radiographic evaluation. acceptable radiographs have non - distorted images of the treated molars and the osseous structures immediately adjacent to the roots. radiographs that did not meet these criteria were excluded from the evaluation. during radiographic evaluation, the following were determined : presence or absence (or reduce in size) of furcation or periapical radiolucencypresence or absence of pathologic internal or external root resorptionpresence or absence of widening in the periodontal membrane. presence or absence (or reduce in size) of furcation or periapical radiolucency presence or absence of pathologic internal or external root resorption presence or absence of widening in the periodontal membrane. statistical analysis was performed between the clinical and radiographic effects of a. sativum oil and those of formocresol in vital pulpotomy in primary teeth using chi - square, and the level of significance was taken at = 0.05. the clinical findings of the primary molars treated with a. sativum oil after 6 months appeared as the following, from the 20 vital pulpotomies ; only two cases had pain after 6 months. they were accompanied by increased mobility (grade ii) and recorded as treatment failure cases. the clinical outcomes of treated primary molars with formocresol after 6 months appeared as the following, from the 20 vital pulpotomies treated with formocresol, only four children had pain that recorded as treatment failure cases and treated by subtotal pulpectomy. comparisons between the clinical findings of vital pulpotomy in primary molars treated with allium sativa oil and those of formocresol are presented in table 1 and graph 1. comparison between the effects of a. sativum oil and those of formocresol on the clinical findings of the first group at 6 months. a histogram showing a comparison between the effects of allium sativum oil (as) and those of formocresol (fc) on the clinical findings at 6 months post - operative. the radiographic outcomes of primary molars treated with a. sativa oil after 6 months showed, from the 20 vital pulpotomies ; there were radiographic changes, only two molars showed evidence of widening in the periodontal membrane space. in addition, two cases exhibited periapical and furcation radiolucency after 6 months and considered as treatment failure cases. moreover, the pericoronal sac associated with the underlying developing permanent teeth showed no noticeable changes (figure 1). mandibular first primary molar showed no changes at 6 months post - operative (b), compared to pre - operative radiograph (a). the radiographic outcomes of primary molars treated with formocresol showed, from the 20 vital pulpotomies ; there were radiographic changes, six primary molars showed evidence of widening in the periodontal membrane space. in addition, four cases exhibited periapical, and two had furcation radiolucency after 6 months and considered as treatment failure cases. furthermore, the teeth showed periapical and furcation radiolucency and considered as treatment failure cases, none of all treated molars exhibited root resorption. in addition, the pericoronal sac associated with the underlying developing permanent teeth showed no noticeable changes (figure 2). mandibular first and second primary molars showed no changes at 6 months post - operative (b) compared to pre - operative radiograph (a). comparisons between the radiographic findings of vital pulpotomy in primary molars treated with a. sativa oil and those of formocresol are presented in table 2 and graph 2. comparison between the effects of a. sativum oil and those of formocresol on the radiographic findings of the first group at 6 months. a histogram showing comparison between the effects of allium sativum oil and those of formocresol on the radiographic findings at 6 months post - operative. statistically, these results revealed no significant difference between the radiographic findings of vital pulpotomy in primary molars with the two medicaments was found. the clinical findings of the primary molars treated with a. sativum oil after 6 months appeared as the following, from the 20 vital pulpotomies ; only two cases had pain after 6 months. they were accompanied by increased mobility (grade ii) and recorded as treatment failure cases. the clinical outcomes of treated primary molars with formocresol after 6 months appeared as the following, from the 20 vital pulpotomies treated with formocresol, only four children had pain that recorded as treatment failure cases and treated by subtotal pulpectomy. comparisons between the clinical findings of vital pulpotomy in primary molars treated with allium sativa oil and those of formocresol are presented in table 1 and graph 1. comparison between the effects of a. sativum oil and those of formocresol on the clinical findings of the first group at 6 months. a histogram showing a comparison between the effects of allium sativum oil (as) and those of formocresol (fc) on the clinical findings at 6 months post - operative. the radiographic outcomes of primary molars treated with a. sativa oil after 6 months showed, from the 20 vital pulpotomies ; there were radiographic changes, only two molars showed evidence of widening in the periodontal membrane space. in addition, two cases exhibited periapical and furcation radiolucency after 6 months and considered as treatment failure cases. moreover, the pericoronal sac associated with the underlying developing permanent teeth showed no noticeable changes (figure 1). mandibular first primary molar showed no changes at 6 months post - operative (b), compared to pre - operative radiograph (a). the radiographic outcomes of primary molars treated with formocresol showed, from the 20 vital pulpotomies ; there were radiographic changes, six primary molars showed evidence of widening in the periodontal membrane space. in addition, four cases exhibited periapical, and two had furcation radiolucency after 6 months and considered as treatment failure cases. furthermore, the teeth showed periapical and furcation radiolucency and considered as treatment failure cases, none of all treated molars exhibited root resorption. in addition, the pericoronal sac associated with the underlying developing permanent teeth showed no noticeable changes (figure 2). mandibular first and second primary molars showed no changes at 6 months post - operative (b) compared to pre - operative radiograph (a). comparisons between the radiographic findings of vital pulpotomy in primary molars treated with a. sativa oil and those of formocresol are presented in table 2 and graph 2. comparison between the effects of a. sativum oil and those of formocresol on the radiographic findings of the first group at 6 months. a histogram showing comparison between the effects of allium sativum oil and those of formocresol on the radiographic findings at 6 months post - operative. statistically, these results revealed no significant difference between the radiographic findings of vital pulpotomy in primary molars with the two medicaments was found. in this study, a comparison between the clinical and radiographic outcomes of a. sativum oil and formocresol as pulpotomy medicaments in primary molars was successfully obtained. since formocresol is still considered the standard in primary teeth pulp therapy, hence it was used in the present study as a control. due to its hazardous effects it produced enamel defects in the permanent successors.15 a systemic uptake of formocresol has been found from pulpotomized teeth, and tissue changes have occurred in various internal organs, particularly in kidney and liver, the quantity of circulating formocresol was found to increase with the number of teeth treated.16 it is known to have toxic, mutagenic and carcinogenic potential (hill., 1991).17 in this regard, a demand for medicament to replace formocresol as a pulp dressing material became imperative. despite the penetration of the modern medicine, traditional medicine continues to be a viable health alternative for the large underprivileged sections of the world.18 hence, increase utilization of indigenous plant medicines in developing countries became a world health organization policy in the 1970s.19 medicinal plants constitute a promising source of several drugs, among the promising medicinal plants, allium sativa, which considered being an amazing herb with a rich historical and religious background.11 in spite of the fact that allium sativa oil was used for the first time in treatment of vital primary molars, the results were remarkable and promising, as none of the succeeded cases showed any signs or symptoms of morbidity during the whole follow - up period. moreover, no obvious radiographic changes were noticed during the recall visits. in the present study, the results revealed that the success rate of a. sativum oil was 90%, while the success rate of formocresol was 85%. in general, these results confirmed the success rate of formocresol in treatment of vital pulps of primary molars and it is in agreement with other studies,20 however, the success rate of formocresol in the present study was lower than that suggested by others. in their study, 100% clinical success rate with formocresol was reported.13 the promising clinical outcome of vital pulpotomy performed with allium sativa oil may be explained on the bases that the oil was found to possess a potent analgesic and anti - inflammatory properties and it is used in the folk medicine for treatment of toothache without any side effects, the analgesic effect might be due to ajoene and diallyl sulfide, which inhibit prostaglandin, the anti - inflammatory effect of allium sativa was reported by several investigators.21 it inhibits prostaglandins through the suppression of cyclooxygenase enzyme in the inflamed area, and inflammatory cytokines.22 it also inhibits prostaglandins through suppression of 5-lioxygenase enzyme. in addition, allicin and ajoene appear to inhibit nitric oxide synthesis in macrophages.23 nitric oxide (a pro - inflammatory mediator) is produced by macrophages.24 in this study, there was a high prevalence of furcation pathosis determined by radiographic examination. this finding is in agreement with another study.25 in the present study, radiographic investigation revealed an improvement, at the end of the follow - up period, with both allium sativa oil and formocresol treated teeth. the clinical and radiographic results revealed success at the aforementioned interval, if the recall periods were continued longer, complete resolution of these changes could be occurred. statistical analysis revealed no significant difference between the effects of both medicaments on the clinical and radiographic outcomes. a. sativum oil offers a good healing potential, leaving the remaining pulp tissue healthy and functioning. statistically, there was no significant difference in the clinical and radiographic success rates of vital pulpotomies in primary molars treated with either a. sativum oil or formocresol.	background : the objective of this study was to compare between the clinical and radiographic effects of allium sativum oil and those of formocresol in vital pulpotomy in primary teeth.materials and methods : a total of 20 children age ranged from 4 to 8 years were included in the study. in every one of those children, the primary molars indicated for pulpotomy. pulpotomy procedure was performed, and the radicular pulp tissue of one molar capped with a. sativum oil in a cotton pellet, whereas the other molar capped with formocresol, the teeth evaluated clinically and radiographically before and after 6 months, using standard clinical and radiographical criteria. statistically, these results revealed no significant difference between the radiographic findings of vital pulpotomy in primary molars with the two medicaments was found.results:a. sativum oil offers a good healing potential, leaving the remaining pulp tissue healthy and functioning. vital pulpotomy with allium sativa oil was given raise 90% success rate while that with formocresol was 85%.conclusion : a. sativum oil is a biocompatible material that is compatible with vital human pulp tissue. it offers a good healing potential, leaving the remaining pulp tissue healthy and functioning.
briefly, the study was conducted at four clinical centers. centers in oakland and los angeles, california, recruited non - hispanic whites and african americans from kaiser permanente, a nonprofit hmo. two other centers in san antonio, texas, and san luis valley, colorado, recruited non - hispanic whites and hispanics from two ongoing population based studies, the san antonio heart study and the san luis valley diabetes study. a total of 1,625 individuals were enrolled (56% women) between october 1992 and april 1994. a follow - up examination was performed 5 years after the baseline examination (range 4.56.6 years). the present report includes information on 826 (79.2%) participants (332 non - hispanic whites, 206 african americans, and 288 hispanics) after excluding 153 individuals who failed to return to the follow - up visit and 64 individuals with missing information on relevant variables. baseline characteristics were similar in the participants included in this analysis and those who were excluded (e.g., age, ethnicity, sex, glucose tolerance status, bmi, waist circumference, and si [all comparisons, p 0.2 ]) except for air (higher in eligible participants, p = 0.005). each examination required two visits, one week apart, of 4 h each. during the first visit, a 75-g oral glucose tolerance test was administered to assess glucose tolerance status. during the second visit, insulin sensitivity and first - phase insulin secretion first, an injection of regular insulin was used to ensure adequate plasma insulin levels for the accurate computation of insulin sensitivity across a broad range of glucose tolerance (20). second, the reduced sampling protocol (12 samples) was used because of the large number of subjects. si and sg were calculated using mathematical modeling methods (minmod, version 3.0, los angeles, california, courtesy of richard bergman, phd) (21). air was calculated as the mean of 2- and 4-min insulin concentrations after glucose administration and di as the product of si and air. plasma glucose and insulin concentrations were determined by the glucose oxidase and dextran - charcoal radioimmunoassay methods, respectively. the 1999 world health organization criteria were used to define diabetes (fasting glucose concentration 7.0 mmol / l, 2-h plasma glucose concentration 11.1 mmol / l, or treatment with hypoglycemic medications) and igt (2-h plasma glucose level between 7.8 and < 11.1 mmol / l). the analysis was carried out using the sas statistical software (version 9.1, sas institute, cary, nc). one - way ancova or logistic regression analysis was used to compare the differences for continuous or dichotomous variables between groups, respectively. linear regression analyses were used to examine the relation of sg to si, air, and di. in separate models risk of future diabetes associated with si, air, di, and sg was assessed by logistic regression analysis. odds ratios (ors) were expressed for binary traits or per sd increase for continuous traits. demographic variables, family history of diabetes, measures of obesity, and plasma glucose concentrations were also included as covariates. we assessed the impact of these covariates on the relation of di and sg to conversion to diabetes by including appropriate interaction terms in separate logistic regression models. in both linear and logistic regression models, log - transformed values of si, air, and di were used to improve discrimination and calibration of the models and to minimize the influence of extreme observations. given that some individuals had si and di equal to zero, we used the natural logarithms of si + 1 and di + 1 as the transformation for si and di, respectively. accuracy for the prediction of diabetes was determined by the area under the receiver operating characteristic curve (auc). the analysis was carried out using the sas statistical software (version 9.1, sas institute, cary, nc). one - way ancova or logistic regression analysis was used to compare the differences for continuous or dichotomous variables between groups, respectively. linear regression analyses were used to examine the relation of sg to si, air, and di. in separate models risk of future diabetes associated with si, air, di, and sg was assessed by logistic regression analysis. odds ratios (ors) were expressed for binary traits or per sd increase for continuous traits. demographic variables, family history of diabetes, measures of obesity, and plasma glucose concentrations were also included as covariates. we assessed the impact of these covariates on the relation of di and sg to conversion to diabetes by including appropriate interaction terms in separate logistic regression models. in both linear and logistic regression models, log - transformed values of si, air, and di were used to improve discrimination and calibration of the models and to minimize the influence of extreme observations. given that some individuals had si and di equal to zero, we used the natural logarithms of si + 1 and di + 1 as the transformation for si and di, respectively. accuracy for the prediction of diabetes was determined by the area under the receiver operating characteristic curve (auc). during the 5-year follow - up period, 128 (15.5%) of 826 iras participants developed type 2 diabetes : 44 (7.9%) of 557 participants with normal glucose tolerance at baseline, and 84 (31.2%) of 269 participants with igt at baseline. those participants converting to type 2 diabetes did not differ from nonconverters in terms of sex or ethnicity (table 1). converters also had higher bmi and waist circumference, higher glucose and insulin levels, and lower si, air, di, and sg. baseline characteristics by diabetes status at follow - up data are n (%) with 95% ci or means sd. results are adjusted for age, sex, race / ethnicity, and research center. these variables were then back - transformed to their units for presentation in the table. in linear regression models, sg was directly related to si (parameter estimate 1 sd, 0.11 0.02, p < 0.001), air (parameter estimate 1 sd, 0.22 0.03, p < 0.001), and di (parameter estimate 1 sd, 0.29 0.05, p < 0.001) after accounting for the effects of age, sex, race / ethnicity, and research center. there was no interaction effect of family history of diabetes on the relation of sg to si (p = 0.534), air (p = 0.139), and di (p = 0.990). we used the auc to quantify the ability of di to predict conversion to diabetes relative to that of a model with both si and air (found in the supplemental figure, available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc10-0165/dc1). the auc of the model with si and air was similar to that of di (0.767 vs. 0.774, p = 0.543). the 5-year incidence of diabetes by baseline tertiles of sg and each one of the other measures (si, air, or di) are presented in fig. 1. five - year incidence of diabetes by tertiles of si, air, di, and sg. results were adjusted for age, sex, race / ethnicity, research center, igt, family history of diabetes, and bmi. cut points for tertiles of si (10 min u ml) were 1.16 (lower), 1.172.38 (middle), and 2.39 (upper). corresponding cut points for tertiles of air (u / ml) were 37.5, 38.075.0, and 75.5 ; di (10 min u ml per u ml) 55.8, 55.9120.0, and 120.1 ; sg (10 ml), 1.58, 1.592.22, and 2.23. sg predicted future development of diabetes after adjusting for age, sex, race / ethnicity, and research center (or 1 sd, 0.50 [0.400.64 ]), as did di (or 1 sd, 0.47 [0.400.56 ]). in a multivariate logistic regression model that included sg and di as independent variables, the odds remained statistically significant for both sg (or 1 sd, 0.61 [0.470.80 ]) and di (or 1 sd, 0.68 [0.540.85 ]) (table 2). age, sex, race / ethnicity, research center, family history of diabetes, fasting glucose, 2-h glucose, bmi, and waist circumference were also included as covariates. in a different model that included si, air, and sg, these three measures were all independent predictors of diabetes. predictors of conversion to type 2 diabetes by multiple logistic regression analysis ors expressed for binary traits or per 1 sd unit change for continuous traits. results in model 1 adjusted also for race / ethnicity (p = 0.494) and research center (p = 0.006) ; log - transformed variables ; results in model 2 also adjusted for race / ethnicity (p = 0.446) and research center (p = 0.006). in separate logistic regression models, we examined the impact of age, sex, race / ethnicity, research center, bmi, glucose tolerance status, family history of diabetes on the relationship between sg (or di), and incident diabetes. none of these variables had a significant impact on the relation of sg and di to conversion to diabetes (p 0.16 for all potential interactions) except for age on the relationship between di and conversion to diabetes (p = 0.038). the strength of the relation of di and sg to conversion to diabetes differed little between categories of age, sex, race / ethnicity, glucose tolerance status, bmi, and family history of diabetes (fig. f). additionally, the interaction term di sg was not statistically significant (p 0.71). di and sg were independent risk factors across different degrees of insulin sensitivity and insulin secretion (fig. risk of developing diabetes associated with di and sg by ethnicity, sex, glucose tolerance status, bmi and age categories, family history of diabetes, and tertiles of si and air. age, sex, race / ethnicity, research center, bmi, igt, family history of diabetes, di, and sg were all included as independent variables in all eight models. log - transformed values of di were used to improve discrimination and calibration of the models and to minimize the influence of extreme observations. in cross - sectional analyses, sg is directly related to insulin sensitivity and first - phase insulin secretion. further in prospective analyses, sg and di were found to be independent predictors of conversion to diabetes similarly across race / ethnic groups, varying states of glucose tolerance, family history of diabetes, and obesity. the predictive power of di is comparable to that of the combination of si and air. expanding a previous analysis by martin. (4), goldfine. (5) examined the risk of conversion to diabetes associated with directly measured di in 155 offspring of parents who both had type 2 diabetes and 181 subjects with normal glucose tolerance and without family history of diabetes. concluded that di and particularly insulin resistance was not sufficient for the development of diabetes in individuals without family history of the disease. these individuals, however, had a very low incidence of diabetes (6 cases during a mean follow - up period of 25 years). in another study of pima indians, incident diabetes was predicted by di independent of the effect of demographic variables and percentage of body fat (7). our results validate previous studies and extend the findings to men and women, all three race / ethnic groups, and varying states of glucose tolerance, family history of diabetes, and adiposity. ferrannini and mari (10) have questioned the hyperbolic paradigm of the relationship between insulin sensitivity and first - phase insulin secretion. in a recent cross - sectional study (22), however, these authors confirmed the paradigm while arguing against the compensation for insulin resistance as the sole mechanism responsible for hyperglycemia. they also indicated that an intrinsic -cell function defect is the most important mechanism for hyperglycemia. in agreement with the hyperbolic paradigm, our results suggest that the predictive power of di is comparable with that of its components. di, however, is not the only independent risk factor for the development of diabetes. since fasting and 2-h plasma glucose values remain strong predictors of diabetes, we can not disregard other determinants of glucose homeostasis, such as additional aspects of secretory response to glucose stimulation (e.g., -cell glucose sensitivity and potentiation), from the disease process. lopez. (23) recently reported that sg is related to si in individuals with family history of diabetes but not in those without such a history. our results, however, indicate that family history of diabetes has little influence on the relationship between sg and si. sg is independent of the dynamic insulin response but is influenced by the basal insulin level (13). in other words, sg is influenced by the effect of insulin on glucose uptake by insulin - dependent tissues (basal insulin similarly, in longitudinal studies by martin. (4) and goldfine. (5), sg predicted conversion to diabetes independent of di but only among offspring of type 2 diabetic parents. individuals without family history of diabetes had a very low risk of future diabetes and neither sg nor di predicted diabetes. (18) examined the risk of progression to igt or diabetes associated with sg in 81 first - degree relatives of african americans with type 2 diabetes. sg as well as directly measured insulin resistance and secretion were independent predictors of worsening glucose tolerance status. nevertheless, none of these studies adjusted their results for glucose tolerance status and adiposity. in the iras, sg is an independent risk factor for future diabetes in individuals with family history of diabetes and similar results are demonstrated in all other categories regardless of age, sex, race / ethnicity, glucose tolerance, and adiposity. experiments in canines have suggested that this relationship is an artifact of the minimal model method (24). in mice, however, the effect of first - phase insulin secretion (in physiological conditions) on sg is minimal (20). furthermore, fsigtt with the minimal model analysis may overestimate sg. in human subjects, the difference between minimal model- and clamp - derived sg appears to be related to the assumption of mono - compartmental distribution of glucose by the minimal model analysis ; however, both measures are highly correlated (25). therefore, minimal model - derived sg is considered an adequate measure of nonsteady - state glucose kinetics and a dependable index. in conclusion, the predictive discrimination of di for future diabetes is comparable with that of the combination of si and air ; therefore, our results support the validity of the hyperbolic paradigm. di and sg are important, independent determinants of diabetes in different ethnic groups and varying states of glucose tolerance, family history of diabetes, and obesity. prospective studies are needed to examine the natural course of sg relative to that of insulin resistance and -cell function.	objectivedisposition index (di) and glucose effectiveness (sg) are risk factors for diabetes. however, the effect of di and sg on future diabetes has not been examined in large epidemiological studies using direct measures.research design and methodsinsulin sensitivity index (si), acute insulin response (air), and sg were measured in 826 participants (aged 4069 years) in the insulin resistance atherosclerosis study (iras) by the frequently sampled intravenous glucose tolerance test. di was expressed as si air. at the 5-year follow - up examination, 128 individuals (15.5%) had developed diabetes.resultsthe area under the receiver operating characteristic curve of a model with si and air was similar to that of di (0.767 vs. 0.774, p = 0.543). in a multivariate logistic regression model that included both di and sg, conversion to diabetes was predicted by both sg (odds ratio 1 sd, 0.61 [0.470.80 ]) and di (0.68 [0.540.85 ]) after adjusting for demographic variables, fasting and 2-h glucose concentrations, family history of diabetes, and measures of obesity. age, sex, race / ethnicity, glucose tolerance status, obesity, and family history of diabetes did not have a significant modifying impact on the relation of sg and di to incident diabetes.conclusionsthe predictive power of di is comparable to that of its components, si and air. sg and di independently predict conversion to diabetes similarly across race / ethnic groups, varying states of glucose tolerance, family history of diabetes, and obesity.
the diagnosis of diffuse interstitial lung disease is performed by a multidisciplinary team that involves pulmonologists, radiologists and pathologists. close collaboration between these different areas of expertise is required to define which patients will need a biopsy and those in whom biopsy is not necessary. surgical pulmonary biopsy continues to be considered the gold standard for recognising histological patterns and aiding the pulmonologist in considering possible causes of disease [2, 3 ]. conventional transbronchial biopsy using forceps (tbb) serves to diagnose sarcoidosis, neoplasias, some infections and cryptogenic organising pneumonia (cop). however, the small size of the samples obtained and the artefacts produced by the forceps on the tissue makes this insufficient in a large number of patients [14 ]. recently, studies have been published that confirm the efficacy of new treatments, such as pirfenidone, in patients with idiopathic pulmonary fibrosis (ipf). the early diagnosis of disease is thus important. with transbronchial cryobiopsies carried out using cryoprobes, the quality of samples obtained and the diagnostic yield are improved compared with transbronchial biopsies with forceps [6, 7 ]. however, the requirement to perform it in an operating room with general anaesthesia and tracheal intubation [810 ] makes it closer to being a surgical technique than conventional tbb performed by a pulmonologist. a prospective study was conducted in the central university hospital of asturias (hospital universitario central de asturias huca) from june 2013 to december 2015 and was approved by the ethics committee of the hospital. patients were referred to the unit of interventional bronchoscopy and pleura of the hospital after being assessed by the multidisciplinary committee on diffuse interstitial lung diseases, which is composed of pulmonologists, radiologists, pathologists and rheumatologists who are experts on autoimmune diseases. we enrolled patients who had clinical and radiographic features of interstitial lung disease (ild) in high - resolution computed tomography (hrct), which was inconsistent with usual interstitial pneumonitis (uip). patients with images typical of uip in hrct according to the british thoracic society guidelines were excluded. cryo - transbronchial lung biopsies (cryo - tbbs) were performed in the most affected lobes according to imaging studies of the chest, following the recommendations of the guidelines [14 ], although always with a preference for the lower lobes for greater safety. the tissue quality variables recorded were tissue sample size (diameter and area), the presence of pleural tissue, and the percentage of artefact - free tissue in each sample. signs of atelectasis, tissue sample fragmentation and clots were considered artefacts ; the pathologists noted these and then recorded how much of each sample was free of such artefacts. the duration times of reintubation of patients airways with the second bronchoscope and the complete time for the procedure were recorded. bleeding was collected into a mucus resorvoir (mocstrap ; proclinics, barcelona, spain), which has a scale in millilitres, and quantified according to the following classification : mild haemorrhage, bleeding 40 ml (table 1). in all patients, the protocol included complete lung function tests, hrct, complete blood count, coagulation and echocardiogram. we excluded patients who presented with blood dyscrasias, severe respiratory failure or unstable heart diseases, anticoagulant medication, pulmonary hypertension, forced expiratory volume in 1 s (fev1) of 40 ml0 (0%)length of procedure min20 (1225)length of reintubation with second bronchoscope s8 (515)number of samples3 (15)diameter mm5.11.8area mm15.79presence of pleural tissue075% artefact - free % of total sampled area75.2%pneumothorax5 (4.7%)data are presented as mean (range) or meansd, unless otherwise stated. rll : right lower lobe ; ml : middle lobe ; lll : left lower lobe ; lul : left upper lobe. : two of the patients had pneumothoraces and required placement of chest tube for 48 h. procedural details of patients who underwent cryo - transbronchial lung biopsy data are presented as mean (range) or meansd, unless otherwise stated. rll : right lower lobe ; ml : middle lobe ; lll : left lower lobe ; lul : left upper lobe. : two of the patients had pneumothoraces and required placement of chest tube for 48 h. all procedures were performed in the bronchoscopy suite with supplemental oxygen delivery through a nasal catheter placed in the larynx above the vocal cords, or sometimes intratracheally (figure 1a). the patient was placed in the anti - trendelenburg position, and continuous records of oxyhaemoglobin saturation, blood pressure, heart rate, respiratory rate and ecg were made. local anaesthesia of the upper and lower airway was performed by instilling lidocaine through a catheter (perifix - braun medical, bethlehem, pa, usa) introduced in the working channel of the bronchoscope (figure 1b). sedation was performed with midazolam (0.07 mgkg) and fentanyl citrate (0.52 gkg), starting with boluses of 13 mg of midazolam and 0.1 g of fentanyl citrate. sedation was maintained with intermittent boluses of 1.2 mg midazolam and 0.1 g fentanyl citrate according to the clinical judgment of the pulmonologist who directed the team of the unit, which was composed of two interventional pulmonologists, two expert nurses and one nursing assistant. two flexible bronchoscopes connected to two different video processors were used (figure 1c). a) nasal catheter placed in the larynx above the vocal cords or sometimes intratracheally. b) instillation of lidocaine through a perifix catheter introduced in the working channel of the bronchoscopy. the first bronchoscope (model eb 1975k, with a 2.8 mm working channel diameter), connected to one of the two processors, was introduced through a short bite block (figure 1d) until the desired segment was located correctly to perform biopsies. the flexible cryoprobe (1.9 mm diameter and 900 mm length ; figure 2a), connected to cryotherapy equipment (erbokryo ca, erbe, germany ; figure 2b), was introduced into the working channel of the bronchoscope, moving forward distally until it was noted that the cryoprobe could not advance further. the cryoprobe was gently retracted by approximately 12 cm, as determined by the marks on the distal area of the probe (figure 2c). with the probe in that position, the erbokryo pedal was pressed for 5 s, freezing the probe point and causing the tissue sample to adhere to the probe. this sample was extracted along with the bronchoscope for thawing and subsequent processing (figure 2d). the second bronchoscope (model eb 1970 tk with a 3.2 mm working channel) was immediately introduced, and was connected to the second processor and prepared for reintroduction into the patient 's airways in a manoeuvre that lasted 510 s and allowed immediate control of any haemorrhage resulting from the cryobiopsy by suction or by instilling substances such as adrenaline, ice - cold saline or amchafibrin. if there were no complications with this second bronchoscope, then we obtained the second sample. the two bronchoscopes were alternated until the desired number of samples (two to five) was obtained. a) the flexible 1.9 mm diameter and 900 mm length cryoprobe, b) connected to the cryotherapy equipment. c) marks on the distal area of the probe (1 cm between each mark). d) cryo - transbronchial lung biopsy samples from one of the patients. when it was confirmed that no bleeding had occurred and that the patient was well controlled, the procedure was stopped. chest radiography or pleural echography was then performed to confirm that a pneumothorax had not taken place, and the patient was discharged 2 h after verifying that there were no complications. follow - up was conducted on all patients at 24 h via phone call to check that there were no delayed complications. all procedures were performed in the bronchoscopy suite with supplemental oxygen delivery through a nasal catheter placed in the larynx above the vocal cords, or sometimes intratracheally (figure 1a). the patient was placed in the anti - trendelenburg position, and continuous records of oxyhaemoglobin saturation, blood pressure, heart rate, respiratory rate and ecg were made. local anaesthesia of the upper and lower airway was performed by instilling lidocaine through a catheter (perifix - braun medical, bethlehem, pa, usa) introduced in the working channel of the bronchoscope (figure 1b). sedation was performed with midazolam (0.07 mgkg) and fentanyl citrate (0.52 gkg), starting with boluses of 13 mg of midazolam and 0.1 g of fentanyl citrate. sedation was maintained with intermittent boluses of 1.2 mg midazolam and 0.1 g fentanyl citrate according to the clinical judgment of the pulmonologist who directed the team of the unit, which was composed of two interventional pulmonologists, two expert nurses and one nursing assistant. two flexible bronchoscopes connected to two different video processors were used (figure 1c). a) nasal catheter placed in the larynx above the vocal cords or sometimes intratracheally. b) instillation of lidocaine through a perifix catheter introduced in the working channel of the bronchoscopy. the first bronchoscope (model eb 1975k, with a 2.8 mm working channel diameter), connected to one of the two processors, was introduced through a short bite block (figure 1d) until the desired segment was located correctly to perform biopsies. the flexible cryoprobe (1.9 mm diameter and 900 mm length ; figure 2a), connected to cryotherapy equipment (erbokryo ca, erbe, germany ; figure 2b), was introduced into the working channel of the bronchoscope, moving forward distally until it was noted that the cryoprobe could not advance further. the cryoprobe was gently retracted by approximately 12 cm, as determined by the marks on the distal area of the probe (figure 2c). with the probe in that position, the erbokryo pedal was pressed for 5 s, freezing the probe point and causing the tissue sample to adhere to the probe. this sample was extracted along with the bronchoscope for thawing and subsequent processing (figure 2d). the second bronchoscope (model eb 1970 tk with a 3.2 mm working channel) was immediately introduced, and was connected to the second processor and prepared for reintroduction into the patient 's airways in a manoeuvre that lasted 510 s and allowed immediate control of any haemorrhage resulting from the cryobiopsy by suction or by instilling substances such as adrenaline, ice - cold saline or amchafibrin. if there were no complications with this second bronchoscope, then we obtained the second sample. the two bronchoscopes were alternated until the desired number of samples (two to five) was obtained. a) the flexible 1.9 mm diameter and 900 mm length cryoprobe, b) connected to the cryotherapy equipment. c) marks on the distal area of the probe (1 cm between each mark). d) cryo - transbronchial lung biopsy samples from one of the patients. when it was confirmed that no bleeding had occurred and that the patient was well controlled, the procedure was stopped. chest radiography or pleural echography was then performed to confirm that a pneumothorax had not taken place, and the patient was discharged 2 h after verifying that there were no complications. follow - up was conducted on all patients at 24 h via phone call to check that there were no delayed complications. during the study period, 106 patients with ild in hrct were included for cryo - tbbs. in all patients, table 2 shows the general characteristics of the patients, while table 1 presents the procedure characteristics. in 37 patients (35%), cryo - tbbs were performed in more than one lobe, and in all patients cryo - tbbs were performed in at least two segments of the same pulmonary lobe. % of reference value ; fev1 : forced expiratory volume in 1 s ; tlc : total lung capacity ; dlco : carbon monoxide diffusing capacity of the lung ; va : alveolar volume. a mild haemorrhage (< 10 ml) occurred in 84% of patients, and 16% had moderate haemorrhage (1040 ml). no patient had a severe haemorrhage that required stopping the procedure and defining special measures, such as admission to the intensive care unit, surgical treatment, transfusions and so on. we had five patients with pneumothorax (4.7%), of which three were resolved with conservative measures without requiring admission and another two required a pleural drainage tube and admission for 48 h. we obtained an average of three biopsies per patient, with an average diameter of 5.1 mm (figure 2d) and a mean specimen area of 15.79 mm, always trying to obtain a representative sample from each segment of the targeted lobe. the average time per procedure was 9.7 min, and the average reintubation time with the second bronchoscope was 8.5 s. in all patients, samples were obtained with a representation of pulmonary parenchyma. a definitive diagnosis after consensus of the multidisciplinary committee was obtained in 91 patients (86%) (table 3), and there was no change in the histopathological diagnoses informed by the pathologists after being assessed by the multidisciplinary committee. in three (2.8%) of the 15 patients in whom transbronchial cryobiopsies were inconclusive, a surgical biopsy (video - assisted thoracoscopic surgery (vats)) was performed, with the final diagnoses being mild interstitial fibrosis, unclassifiable interstitial pneumonia, and desquamative interstitial pneumonitis (the patient had a prolonged air leak as a complication of surgery). in the 12 remaining patients, cryobiopsy served to rule out other processes such as neoplasia, infection, granulomatosis or drug toxicity, and it was decided to conduct conservative follow - up as surgical biopsy was contraindicated by comorbidities and the increased risk they presented. the most common diagnoses of our series were as follows : idiopathic pulmonary fibrosis (ipf / uip) (n=22), cryptogenic obstructive pneumonia (cop) (n=10), nonspecific interstitial pneumonia (nsip) (n=11), and silicosis (n=12). the pathological anatomy of a desquamative pneumonitis (figure 3a c) and a pneumoconiosis (figure 3d f), common in our geographic area, are shown. final diagnosis after multidisciplinary review data are presented as number of subjects. a 45-year - old smoking man diagnosed with desquamative interstitial pneumonia (dip) by a) cryo - transbronchial lung biopsy (haematoxylin eosin stain) showing b) thick alveolar septa filled by numerous macrophages with c) cd68 staining. a 67-year - old man diagnosed with silicosis by d) cryo - transbronchial lung biopsy showing e) a silicotic nodule (black arrow) and f) silica crystals with polarised light (white arrow). we have demonstrated that the cryo - tbb used for the diagnosis of ild can be performed in an adequately prepared bronchoscopy suite equipped with the necessary means without needing to intubate the patient, without fluoroscopy and with moderate sedation. this procedure consists of using two flexible bronchoscopes connected to two video processors that we alternated by the oral route until obtaining the desired number of samples. the alternation of two bronchoscopes by the oral route permits the procedure to be rapid and allows for any haemorrhages produced by cryobiopsy to be controlled immediately, because we are able to reintubate the patient within a few seconds. when this is conducted by a single bronchoscope through the nose, the time that it remains outside the airway is greater than 1 min, which is how long the cyroprobe takes to thaw and release the cryobiopsy adhered to it. if a haemorrhage were produced, we would not be able to control it immediately. in addition, the cryobiopsy could be released upon removing the bronchoscope along the narrow nasal passage and thus lost. although our method allows for rapid control of the patient 's airway, our greatest concern was always the possible occurrence of complications, especially haemorrhage. to prevent haemorrhage, we selected patients by rigorously excluding those who had a greater risk of having haemorrhages (blood dyscrasias, anticoagulant medication, pulmonary hypertension, among others) and we attempted to perform cryobiopsies in the lower lobes if pulmonary involvement on the hrct was diffuse, due to the greater time a bronchoscopist has to control a possible haemorrhage in this location than in the upper lobes. as our learning curve progressed, we also included the upper lobes when the more affected lobe in hrct was located there, always placing the patient in the anti - trendelenburg position during the entire procedure. using this method, haemorrhage in our series was mild or moderate in all patients, and we controlled bleeding with suction, adrenaline, ice - cold saline and amchafibrin. in we thus believe that the appropriate selection of patients, the experience of the interventional pulmonologist who performed the procedure and of the team that formed the unit, and the haemostasis effect produced by freezing the probe on the tissue are key to our success. it is also important to emphasise that the number of pneumothoraces in our study is lower than in other published studies performed in an operating room under general anaesthesia [9, 10 ] and the yield was better [8, 9 ]. our study differs from other studies [610 ] in that we did not use a fluoroscopic guide to place the cryoprobe in the small - calibre airway, making our suite radiation - free. our results regarding cost - effectiveness, complications and lack of pleura in any of the biopsies demonstrate that fluoroscopy is not essential and is most probably unnecessary for this technique. most of the studies published on transbronchial [610 ] or endobronchial cryobiopsies have been performed on intubated patients because the tissue adhered to the cryoprobe can not be extracted through the working channel of the video bronchoscope and because the bronchoscope and cryoprobe need to be removed as a unit. under these conditions, it is important to achieve rapid reinsertion of the bronchoscope to control any possible bleeding produced by the cryoprobe, by suction, tamponade, instillation of substances such as adrenaline, ice - cold saline or even an occlusion balloon. our method allows us to perform all of these manoeuvres in a few seconds, so that the patient 's safety is ensured. the procedure we describe has also allowed us to perform cryobiopsies in endobronchial lesions, with better yield than conventional biopsy with forceps and the same safety, but with the advantage of not having to perform it in an operating room with the patient intubated and under general anaesthesia, which would considerably increase the economic cost. another advantage of this procedure is that, by not needing general anaesthesia or an operating room, and by performing the procedure in the bronchoscopy suite like other procedures we perform in routine clinical practice, after a training period this technique can be performed by pulmonologists who work in hospitals of different levels of care, greatly amplifying the field of future users of the technique. there have been in vitro studies in animals demonstrating that the sample size depends on variables such as tissue type, probe diameter, application time and pressure exerted by the probe on the tissue. these studies have shown that even the smallest probes currently used provide larger biopsies than those obtained with a conventional biopsy forcep, as used in most bronchoscopy suites at present., very recently, have published a study in animals that uses a mini probe to obtain samples through the working channel of a bronchoscope without needing to remove the bronchoscope from the central airway. in our experience, the 1.9 mm diameter probe has provided us with good yield and very few complications. it has a long half - life and it is the probe we advise using at this time, although it would be interesting to conduct comparative studies of the different probes that exist on the market. to date, the debate on how to best perform the procedure is focused on two factors, the type of sedation and control of the airway ; it can be performed with the patient intubated with an endotracheal tube or a rigid tracheoscope under deep sedation or without intubating the patient with conscious sedation and in the bronchoscopy suite. a limitation of our study could be that the patients are in an initial phase of their disease, as can be observed by the lung function values shown in table 2. it is possible that, in more advanced phases of disease (e.g. fev1 < 40%), the yield and complications of the technique would be different, and we therefore believe that new prospective studies are necessary to standardise all aspects of the procedure. we conclude that, in well - selected patients with good lung function and without associated risk factors, our procedure is the first choice due to its many advantages and few drawbacks. cryo - tbb following our method is a minimally invasive, rapid, safe and economic technique that can be performed in a bronchoscopy suite under moderate sedation without the need for intubating the patient or using fluoroscopy, and without requiring general anaesthesia. we conclude that, in well - selected patients with good lung function and without associated risk factors, our procedure is the first choice due to its many advantages and few drawbacks. cryo - tbb following our method is a minimally invasive, rapid, safe and economic technique that can be performed in a bronchoscopy suite under moderate sedation without the need for intubating the patient or using fluoroscopy, and without requiring general anaesthesia.	transbronchial biopsy using forceps (tbb) is the first diagnostic technique performed on patients with interstitial lung disease (ild). however, the small size of the samples and the presence of artefacts in the tissue obtained make the yield variable.our objectives were 1) to attempt to reproduce transbronchial cryobiopsy under the same conditions with which we performed conventional tbb, that is, in the bronchoscopy unit without intubating the patient and without fluoroscopy or general anaesthesia ; 2) to describe the method used for its execution ; and 3) to analyse the diagnostic yield and its complications.we carried out a prospective study that included 106 patients with clinical and radiological features suggestive of ild who underwent cryo - transbronchial lung biopsy (cryo - tbb) under moderate sedation without endotracheal intubation, general anaesthesia or use of fluoroscopy. we performed the procedure using two flexible bronchoscopes connected to two video processors, which we alternated until obtaining the number of desired samples.a definitive diagnosis was obtained in 91 patients (86%). as for complications, there were five pneumothoraces (4.7%) and in no case was there severe haemorrhage or exacerbation of the underlying interstitial disease. cryo - tbb following our method is a minimally invasive, rapid, safe and economic technique that can be performed in a bronchoscopy suite under moderate sedation without the need for intubating the patient or using fluoroscopy and without requiring general anaesthesia.
mangroves are coastal ecosystems that have been seriously threatened by anthropogenic activities. worldwide, mangrove areas have been used for urban development, tourism, oil exploitation, agriculture, and shrimp farming. between 1980 and 2005, about 3.6 million hectares of mangrove was lost. competition for land is the major cause of devastation and losses over time. brazil, the second largest mangrove area in the world, has lost approximately 50,000 ha of mangroves in the last 25 years. although these ecosystems are well known for their typical flora and associated fauna, comparatively, only a few studies deal with their microbial diversity [38 ]. on the other hand, studies on cultivable microorganisms have advanced in the isolation and identification of organisms capable of degrading xenobiotics, including oil hydrocarbons [3, 5, 911 ]. in the environment microorganisms fulfill various niches and are fundamental for the functioning of mangroves, being particularly important in controlling the geochemistry of these habitats [12, 13 ]. recently, using metagenomics and pyrosequencing andreote. retrieved a large volume of information on the microbial composition and function in tropical mangroves. although these studies represent a valuable contribution to our understanding of microbial life, more studies are necessary to access the microbial ecology of mangrove sediments from distinct zones inside the mangrove, as well as those submitted to different anthropogenic threats. genetic fingerprinting techniques provide a pattern or profile of the genetic diversity in a microbial community, which are important in distinguishing pcr products that have different nucleotide sequences. terminal - restriction fragment length polymorphism (t - rflp) has proven to be a valuable tool to study bacterial community structure in complex environments such as sediments and soils [3, 18, 19 ]. also, tools for web - based phylogenetic alignment exist that allow the retrieval of hypothetical microbial diversity. these in silico methods, such as the resources available on the mica3 (microbial community analysis iii) website, make the identification of specific organisms in a community based on the length of terminal - restriction fragments (t - rfs) possible, as they predict t - rfs from known and deposited sequences in databases that can be compared with the submitted t - rfs [20, 21 ]. in this context, we hypothesized that the zonation of mangrove species, as well as the daily fluctuations imposed by the tidal regimes, shapes the microbial communities that are present in these habitats, making them unique to each mangrove area. in order to test our hypothesis, we employed t - rflp to access the composition and structure of bacterial communities of sediments in vegetated and nonvegetated areas in a mangrove located in northeastern brazil and its relation to the biotic and abiotic variables in a region known for petroleum exploitation, which is considered a risk area for oil contamination. barra grande mangrove is located in icapu, on the extreme east coast of the state of cear, northeastern brazil (3720w 440s), in a region comprised of an extensive tidal flat, covering an area of 1,260.31 ha (figure 1). due to the rather flat profile of the studied area, the sampling sites remain uncovered at low tide (0.1 m) and are subsequently flooded by the tide. sediments from three different sites at depths between 0 and 10 cm were collected, following the shoreline in a perpendicular transect. site 1 (s1) was the closest to the sea in an area without vegetation ; the second site (s2) was located in an area of avicennia schaueriana forest ; and the third site (s3) was located in a region of a robust forest of rhizophora mangle. at each site, five sediment samples (010 cm depth) were randomly collected using a cylindrical sampler (30 cm long and 10 cm in diameter) and transferred to sterile jars. the samples were kept in an ice - cooled box for about 2 hours before being transported to the laboratory. in the laboratory, the five replicate samples from each site were homogenized in order to obtain composed and representative samples of each habitat and a portion was stored at 20c for dna extraction and the remaining fraction was used for sediment analyses. granulometry was performed by dry sieving, and organic matter was determined by weight loss on ignition, described in schulte and hopkins. the environmental variables ph, salinity, and temperature of the sediments ' percolated water were measured directly in the field, using a multiparameter probe (multiparameter display system model 650, ysi, yellow springs, oh, usa). bacterial communities were analyzed by t - rflp, following the protocol described by marsh. the extraction of total dna from sediment samples was performed using the powersoil dna isolation kit (mo bio laboratories, carlsbad, ca, usa), following the manufacturers ' protocol. dna samples were amplified by pcr using the primers 63f labeled with the fluorophore 6-carboxyfluorescein (6-fam) at the 5 end and 1389r. pcrs were performed according to the following program : initial denaturation at 94c for 3 min, 25 cycles of 94c for 1 min, 55c for 1 min, 72c for 2 min, and a final extension at 72c for 10 min. pcr products were purified using the commercial kit qiaquick pcr purification (qiagen, valencia, ca, usa). afterwards, samples were digested separately with restriction enzymes hhai and mspi following the manufacturers ' recommendations (new england biolabs, beverly, ma, usa). digestion products were dried at 40c and sent to the research technology support facility, department of plant biology, michigan state university (msu, east lansing, mi, usa) facility, where t - rf profiles were generated. the analysis was performed using 2 l of the digestion with 8 l of a solution containing the internal standard mapmaker 1000 (bioventures inc., murfreesboro, tn, usa) labeled with rox (6-carboxy - x - rhodamine) and the running buffer (deionized formamide). dna fragments were detected by capillary electrophoresis on an abi prism 3100 genetic analyzer (applied biosystems, foster city, ca, usa) automatic sequencer. the t - rfs were visualized using the genescan analysis software (applied biosystems), exported to excel, and analyzed with the ibest tool (http://mica.ibest.uidaho.edu/) using the height of 50 units of fluorescence as an initial point for the electropherogram analysis and normalized by calculating the relative abundance of each t - rf from the fluorescence intensity area. t - rfs in the range of 50990 bp were used for the analysis. the files were exported from ibest and analyzed by the t - align tool (http:/inismor.ucd.ie/~talign / index.html). the relative abundance of otus was used to calculate diversity indices for each sample. the shannon index (h) by log2, the simpson diversity (), and the pielou equitability (j) were calculated using the program primer 6 (primer e, ivybridge, united kingdom). the web - based analysis tool (pat+) provided by mica3 (http://mica.ibest.uidaho.edu/pat.php) was used to identify otus for t - rf peaks, based on the rdp (ribosomal database project) release 9.60 16s rrna gene database. habitats s1 and s2 were relatively similar in most environmental factors, except for the presence of vegetation in s2, and salinity was the only analyzed variable shared between s2 and s3, apart from the presence of vegetation. sediments were classified as fine sand at s1 and s2 and coarse silt at s3, the latter presenting a higher silt + clay and organic matter content. the measured environmental variables temperature, ph, salinity, organic matter content, and sediment particle size from the three habitats of the barra grande mangrove are shown in table 1. three different community structures with a higher similarity between s2 and s3 were observed, both inside forested areas. the digestion with hhai generated a larger number of otus (120 t - rfs) than mspi (87 t - rfs). the relative abundance of otus (figure 2) revealed that s1 had a lower richness (34 t - rfs) and a great abundance of three otus. s3 showed the highest number of t - rfs (73) but a lower relative abundance and was considered the most diverse site in terms of bacterial otus. s2 showed intermediate characteristics when compared with the other sites, showing some abundant t - rfs and also an intermediate number of otus (43). only two t - rfs were identical among the sites as shown in figure 3. in addition, s1 shares only three otus with s2 and s3, whereas s2 and s3 share 20 otus. therefore, s1 showed the highest percentage of unique otus (76.5%), followed by s3 (65.75%) and s2 (41.86%). the comparison of diversity indices (table 2) showed an increase in terms of evenness, richness, and diversity from s1 to s3. using pat+ in mica, we predicted the potential bacterial groups based on digestion pattern of the fragments obtained by t - rflp. t - rfs 54 and 65, which were shared by the three sites, were mainly represented by uncultured bacteria of the bacteroidetes and proteobacteria phyla. among the possible species that can be attributed to t - rf 54, capnocytophaga sp., vibrio sp., and photobacterium sp., whereas many species of bacteroidetes from the flavobacteriaceae family and some alphaproteobacteria were assigned to fragment 65. considering the dominant fragments at each site, s1 showed three different fragments : t - rf 100 associated with uncultured halophilic bacteria ; t - rf 325 represented by uncultured bacteria, including species of gammaproteobacteria and the cultured bacterium vibrio parahaemolyticus ; and t - rf 394 corresponding to an uncultured bacterium. at s2, four dominant fragments were detected : t - rf 57 including alphaproteobacteria such as uncultured azospirillum sp. and the bacteroidetes mariniflexile fucanivorans and cultured and uncultured cytophaga spp. ; t - rf 56 comprising alphaproteobacteria as sneathiella sp., uncultured mesorhizobium sp., various species of thalassospira, members of rhodospirillaceae, some uncultured gammaproteobacteria from piscirickettsiaceae, and groups of the phylum bacteroidetes, such as fluviicola sp., aequorivita sp., a. antarctica, a. sublithincola, subsaxibacter sp., persicivirga sp., salinimicrobium sp., mariniflexile gromovii, myroides sp., m. odoratimimus, m. profundi, m. pelagicus, gelidibacter sp., g. algens, and an uncultured sphingobacterium ; t - rf 77 which could not be identified by the web - based tool ; and t - rf 94 which was identified as escherichia coli. s3 showed three dominant fragments : t - rf 55 represented by uncultured bacteria and capnocytophaga sp. ; t - rf 72 represented by uncultured members of the order bacteroidales ; t - rf 167 which consisted of undetermined uncultured bacteria and several uncultured gammaproteobacteria and cultured representatives such as pseudomonas sp., in this study, we observed differences in the bacterial community structure and composition that could be attributed to the specific characteristics of each sampled mangrove habitat. it is well known that mangroves are under the influence of marine and terrestrial environments, which generate gradients in the texture of sediments and organic matter content and in salinity as a result of sea and freshwater inputs [27, 28 ]. taking this into consideration, it is expected that the fluctuating environmental conditions shape the microbial communities in mangroves. peixoto., 2011, have shown that mangrove microbial communities are heterogeneously distributed within mangroves and between different mangroves. the authors explain these differences based on the sharp environmental gradients over short spatial scales that include pollutants, reductive - oxidative balance (redox state), ph, and nutrient distribution. also, microbial populations seem to be influenced by the presence and type of mangrove species. 2010, demonstrated that, even under the fluctuating conditions found in mangroves, the rhizosphere effect, which is well described for terrestrial plants, was also evidenced in this ecosystem. 2016, found a predominance of bacteroidetes in the rhizosphere of avicennia, while a predominance of actinobacteria was evidenced in nonvegetated sediments.. studying culturable populations showed that the species in the laguncularia rhizosphere harbored the highest microbial population when compared to other mangrove species. sites s2 and s3, which are habitats covered by a. schaueriana and r. mangle, respectively, shared more similarities in terms of microbial composition with each other than with s1, the site without vegetation. the numbers of otus detected in s1, s2, and s3 were 34, 43, and 73, respectively. this increase in the richness from s1 to s3 was observed, which suggests that, besides local abiotic variables, the nature of exudates and nutrients provided by each plant species select specific communities. the observed differences in community structure were reflected in the relative abundance as well as in t - rf composition. regarding the percentage of unique t - rfs, it is notable that this mangrove harbors quite different bacterial communities, as confirmed by the high value of exclusivity, especially at s1, which was 76.5%, and the low level of similarity among the sites, considering that only two t - rfs were shared by them. we detected an increase in the evenness of bacterial communities over the three sites ; that is, s1 showed lower evenness and the presence of some dominant otus. due to the proximity of the sea, the microbiota in s1 is under the influence of tidal hydrodynamics, which probably led to the selection of several species that are more adapted to marine environments. at s3, a wider distribution of otus was observed, with a lower occurrence of dominant otus, demonstrating that the environmental conditions have not favored any particular otu. intermediate characteristics were shown at s2, which can be explained by its location in an area inside the vegetation (a. schaueriana) like s3, but with many abiotic variables similar to those found at s1, due to its proximity to the sea. putative community compositions were determined using a phylogenetic assignment tool (pat) developed by kent., using mica3, in which the sizes of t - rf peaks in mangrove soils were matched with t - rf sizes derived in silico from the 16s rrna gene sequences of phylotypes in the rdp database. the results from pat were used to examine the bacterial community composition at different levels of phylogenetic resolution. at the three studied habitats (s1, s2, and s3), there was prevalence of uncultured bacteria, which shows the wide gap in extant data on microbial diversity, considering the large number of unknown organisms. taking into account the possible groups associated with t - rfs, it can be observed that, besides the uncultured bacteria, the main common otus were phylogenetically affiliated with the bacteroidetes, with a large number belonging to the flavobacteriaceae. in brazilian mangrove sediments, some bacteria affiliated with the bacteroidetes were also observed in clone libraries, but at a low number compared to the phylum proteobacteria, which appears to be dominant in these environments [8, 33, 34 ]. considering the dominant groups at each habitat in the studied mangrove, an overall abundance of proteobacteria and bacteroidetes was observed. at site s1, which lies in a region close to the sea, without vegetation, at s2, located in the root zones of a. schaueriana, several uncultured bacteria were found, with a predominance of alphaproteobacteria, some of them known nitrogen fixers, such as an uncultured azospirillum, which was previously reported in roots of a. marina and in the rhizosphere of suaeda monoecious in a mangrove from india. several bacterial strains affiliated with the genus thalassospira were detected, which were previously isolated from oil - contaminated seawater. it was found that these could degrade several polycyclic aromatic hydrocarbons (pahs), including naphthalene, dibenzothiophene, phenanthrene, and fluorene. these strains might play important roles in the bioremediation of marine oil spills and considering the location of the studied mangrove in a risk area for oil contamination, the presence of possible oil degraders indicates the potential of this environment to respond effectively to possible contamination by petroleum hydrocarbons. among the otus found at s3, some were identified as belonging to the phylum proteobacteria, such as vibrio sp. and photobacterium (proteobacteria) and rhodovulum sulfidophilum, a marine photosynthetic bacterium (alphaproteobacteria). some members of the genus pseudomonas which are able to metabolize petroleum hydrocarbons were also detected. altogether, the data showed distinct bacterial communities among the three mangrove habitats due to the presence and type of vegetation and the divergence environmental variables in which these habitats are submitted. in general, all the potential bacteria corresponding to the identified t - rfs are typical of marine environments and play important roles in maintaining the dynamic balance of the ecosystem. this study highlights the importance of preserving mangrove ecosystems as a whole, due to the uniqueness of each habitat. the main contribution of this study was to demonstrate that mangrove soils hold highly diverse bacterial populations with increasing richness from the sea to forested areas, selected by the local differences. the existence of unique bacterial communities to each mangrove habitat covered by distinct plant species clearly demonstrates the importance of the conservation of this ecosystem as a whole.	we investigated the relationship among environmental variables, composition, and structure of bacterial communities in different habitats in a mangrove located nearby to an oil exploitation area, aiming to retrieve the natural pattern of bacterial communities in this ecosystem. the t - rflp analysis showed a high diversity of bacterial populations and an increase in the bacterial richness from habitats closer to the sea and without vegetation (s1) to habitats covered by avicennia schaueriana (s2) and rhizophora mangle (s3). environmental variables in s1 and s2 were more similar than in s3 ; however, when comparing the bacterial compositions, s2 and s3 shared more otus between them, suggesting that the presence of vegetation is an important factor in shaping these bacterial communities. in silico analyses of the fragments revealed a high diversity of the class gammaproteobacteria in the 3 sites, although in general they presented quite different bacterial composition, which is probably shaped by the specificities of each habitat. this study shows that microhabitats inside of a mangrove ecosystem harbor diverse and distinct microbiota, reinforcing the need to conserve these ecosystems as a whole.
basal metabolic rate (bmr) quantifies the minimum rate of energy expenditure necessary to maintain energy balance of resting, post - absorptive endotherms at thermoneutral conditions (schmidt - nielsen 1997). although conceived with biomedical research in mind, bmr has quickly become the most widely used benchmark metric of metabolic rate (white and kearney 2012). it has also become clear that the composition and variation in bmr convey extremely important biological information (for an extensive review see mcnab 2002). consequently, disentangling the factors and mechanisms that underlie differences in bmr at inter- and intra - specific level has become the key component of major questions at the interface between evolution, ecology, and physiology of endotherms, including the evolution of endothermy itself (hayes 2010 ; nespolo. historically, studies on variation in bmr were guided by the krogh principle (1916), taking advantage of the several - fold inter - specific variation in bmr (mcnab 2002). although inter - specific studies have greatly contributed to our understanding of the general patterns of bmr variation, they are limited by two important factors. first, studies at inter - specific level must take into account the potentially confounding effect of phylogeny on the inference (garland. second, inter - specific analyses are based on the assumption that a species - specific average bmr value used in those analyses adequately characterizes bmr at the intra - specific level. this assumption is legitimate, when inter - specific variation widely exceeds intra - specific variation (ives. it must be borne in mind, however, that it is intra - specific variation that is a substrate of natural selection, and therefore, inter - specific studies on bmr can only partially inform the inference on adaptation and can not unambiguously identify factors influencing its variation. for this reason, patterns derived from inter - specific analyses can not be directly extrapolated to the intra - specific level. on the other hand, however, intra - specific studies on metabolic traits also have their limitations. the most important one is the narrow range of within - species variation, which hampers the power of statistical analysis (konarzewski. this limitation is exactly why researchers often resort to inter - specific comparisons, even though they do not always provide sufficient methodological justification for the inferences they make (garland and adolph 1994). despite the limited statistical power, studies on intra - specific variation in bmr become increasingly attractive thanks to the wealth of information on the molecular underpinnings of energy expenditure, which by definition are more applicable at the intra- than the inter - specific level of inference (stapley. also, the state - of - the - art equipment used to quantify metabolic rates now offers the possibility to reduce the measurement error of bmr down to 15 % (konarzewski. 2005, for an extensive review of measurement techniques see lighton 2008), which most likely underlies the increasing number of studies reporting statistically significant within - species repeatability of bmr (for review see nespolo and franco 2007). here, we decompose variation in bmr and review the literature pertaining to different levels of biological organization that contribute to variation in bmr. in doing so we have exclusively focused on body mass - corrected bmr, because whole - body bmr and its relation to body mass have been recently reviewed (white and kearney 2012). bmrs discussed herein have been analysed by one of general linear models (most often by ancova with whole - body mass as a covariate), which effectively accounts for the effect of body mass and allows for a direct comparison of individuals of different masses. following intense debate (packard and boardman 1987 ; tracy and sugar 1989 ; jasienski and bazzaz 1999) such statistical means of correction of body mass have become widely accepted by integrative physiologists. it is important to note in this context, that the controversy regarding how best to control for the impact of body mass on physiological traits still remains unsettled in biomedical literature (kaiyala and schwartz 2011). also, numerous studies (e.g. szafraska. 2007) do not meet the criteria that animals are quantified in a post - absorptive state, which is part of the definition of bmr. however, as meeting this condition most likely does not appreciably affect the determinants of bmr discussed in this review (e.g. the contribution of organ sizes to bmr), we equate resting metabolic rate (rmr) to bmr. we mainly base our review on mammalian studies, as the majority of the relevant information presented herein comes from research on laboratory rodents and humans. however, wherever possible, we also highlight how the results of laboratory studies can be applied to free - ranging animals, though we do not extensively discuss bmr in an ecological context, as it has been recently reviewed elsewhere (burton. finally, we review recent advancements in the quantitative genetics of bmr and organ mass, as well as the molecular genetics of bmr. at its most fundamental level, whole - body bmr is the sum of the products of organ masses and their mass - specific metabolic rates (schmidt - nielsen 1984 ; wang. rodents, in particular laboratory mice and rats, are undoubtedly the best studied animal models with regard to both of these components, and consequently, their contribution to bmr. visceral organs (heart, kidney, liver, and small intestine) and the brain that are primarily responsible for energy flux comprise ~58 % of body mass of laboratory rats and mice, as well as humans (mller. konarzewski and diamond (1995) tested the intra - specific correlation between bmr and the masses of four visceral organs (heart, kidney, liver, and small intestine) among six inbred strains of laboratory mice. they found that strains with exceptionally high (or low) bmr tended to have disproportionately large (or small) organs. likewise, sacher and duffy (1979) found a positive correlation between bmr and brain mass in laboratory mice. however, several studies carried out on an outbred mf1 strain of laboratory mice have not found significant correlations between bmr and internal organ masses (johnson. such correlations were also absent in studies carried out on several wild rodents (e.g. white - footed mouse, koteja 1996 ; leaf - eared mouse, nespolo. this research builds upon the quickly advancing development of imaging techniques, particularly computer tomography and magnetic resonance imaging (mri), allowing for the quantification of the organ size in vivo and their contribution to variation in human bmr (e.g. later. 2008 ; javed. 2010 ; mller. 2002, 2011). for example, elia (1992) estimated the mass - specific metabolic rates (in kcal / kg per day) of major human organs of young adults to be : 200 for liver, 240 for brain, 440 for heart and kidneys, 13 for skeletal muscle, 4.5 for adipose tissue and 12 for residual mass. these estimates were recently validated with the use of imaging technologies, which also allowed for the fine - tuning of elia s estimates with respect to the effect of gender differences (wang. likewise, a recent imaging - based comprehensive analysis of the scaling of human bmr and organ masses revealed that muscle, brain and liver explained up to 43 % of the inter - individual variance in human bmr (mller. theoretically the most robust test of the existence of a positive association between bmr and metabolically expensive organ masses should be provided by artificial selection experiments aimed at either bmr or the masses of those organs. assuming that there exists sufficient additive genetic variation, such experiments allow for the change of frequencies of alleles directly related to energy expenditures (either at the level of the mass - specific metabolic rates or whole organs). the major advantage of an experimental manipulation of bmr is its explanatory ability to distinguish non - causative correlations between bmr and anatomy from biologically meaningful, inescapable links underlined by the genetic architecture of postulated associations (for an extensive review of artificial experiments on rodents see swallow. if the postulated, inexorable link between bmr and organ masses exists, then such selection should result in concerted unidirectional changes in both directly selected and secondary (correlated) traits (for theoretical justification see hayes 2010). several artificial selection experiments on rodents have achieved substantial changes in bmr (ksiek. 2004) and other metabolic traits including maximum metabolic rate (middleton. 2008 ; wone. 2009), body mass - corrected food intake (bunger. we summarized their major findings in table 1. among these experiments only one directly selected on bmr, resulting in a conspicuous 40 % difference in bmr (quantified as residuals from a regression of bmr on body mass) between low and high selected line types, derived from swiss webster outbred strain of laboratory house mice (ksiek. 2004, 2009 ; konarzewski. 2005 ; brzk. 2007 ; gbczyski and konarzewski 2009 ; for review see swallow. 2009). this between - line difference in bmr was also reflected in considerable differences in internal organ masses : mice from the high - bmr line had larger hearts, livers, kidneys, and small intestines (ksiek. 2004 ; ksiek and konarzewski 2012). those differences between the organ sizes in high and low line types ranged from 14 % for hearts, 17 % for livers, 18 % for kidneys and 13 % for small intestines in generation f19, and increased to 16, 18, 31 and 34 %, respectively, in generation f31. the resulting differences were significantly larger than divergences that could result from random genetic drift alone, and thus support the existence of a genuine genetic correlation between organ masses and bmr.table 1summary of the responses to artificial selection on metabolic and related traits in rodentsselection criterion / method / speciesbmr responsecorrelated traitstrait responsereferencemass - corrected bmr / indirect calorimetry / laboratory mice (mus musculus)increasefood consumptionincreaseksiek. (2009)voluntary activityincreasegbczyski and konarzewski (2009)vo2max (treadmill)no changevo2max (swim elicited)decreaseksiek. (2007)core body temperatureno changegbczyski (2008)brzk. (2012)mass of heart, liver, kidney, (2003)immune response (klh)increaseksiek and konarzewski (2012)mass of spleen and lymph nodesincreasethymus massdecreaseoxidative enzyme capacityincreaseksiek. (2007)mass - corrected food intake / laboratory mice (mus musculus)increasedigestive efficiencyincreasehastings. (2005)liver mass (dry)increasesmall intestine length (fresh)increasesmall intestine mass (dry)no changeselman. (2001a, b)large intestine mass (dry)decreasepancreas mass (dry)no changestomach mass (dry)increasekidneys mass (dry)no changeheart mass (dry)increaselung mass (dry)no changebrain mass (dry)increasethyroid mass (dry)decreasespleen mass (dry)no changeheat loss/(body mass)/direct calorimetry / laboratory mice (mus musculus)not measuredfood consumptionincreasenielsen. (1997b)voluntary locomotor activityincreasenielsen. (1997a)mass of liver, heart, spleenincreasemoody. (1999)core body temperatureincreasemousel. (2001)t4 leveldecreasekgwatalala and nielsen (2004)t3 levelno changecorticosterone levelincreaseexpression of ucp-1decreasemcdaneld. (2002)mass - corrected vo2max / swimming / laboratory mice (mus musculus)no changeheart massincreasegbczyski and konarzewski (2009)mass of liver, kidney, small intestineno changemass of gastrocnemiusincreaseaerobic endurance capacity / treadmill running / rats (rattus norvegicus)not measuredbody massdecreasekoch and britton (2001)fat massdecreasekirkton. (2010)cardiac outputincreasepulmonary functionincreasehowlett. (2003)oxidative enzyme capacityincreaseleft ventricular cells systolic and diastolic functionincreasesmall intestine lengthdecreasewislff. 2005)voluntary locomotor activity / daily wheel running activity / laboratory mice (mus musculus)no changevo2maxincreaseswallow. (2008)heart (ventricle), spleen, liver, adrenal glandsno changeswallow. (2009)three - way selection / bank vole myodes (clethrionomys) glareolusvo2max (swim elicited)increasefood consumptionincreasesadowska. koteja, unpublishedability to grow on a low - quality herbivorous dietno changeintensity of predatory behaviourno changemass - corrected vo2max / treadmill running / laboratory mice (mus musculus)increasethe liver amino acids and tricarboxylic acid cycle (tca cycle) metabolitesdecreasewone. (2011)gastrocnemius, amino acids and tca cycle metabolitesincrease summary of the responses to artificial selection on metabolic and related traits in rodents the results of direct selection on bmr are complementary to those of other artificial selection experiments, which have targeted traits closely correlated with bmr. selman. (2001a, b) showed that artificial selection of laboratory mice for a high rate of food consumption resulted in both larger sizes of the internal organs and bmr, compared with mice from lines selected for a low rate of food consumption (table 1). nielsen. (1997a, b) selected laboratory mice for high and low heat loss, measured in adult males during a 15-h assay using a direct calorimetry system. this selection too resulted in larger metabolically active organs (liver and heart) in line types selected for high heat loss, as compared to the control line types (kgwatalala and nielsen 2004). overall, the results of artificial selection experiments provide mounting evidence for the existence of a strong genetic correlation between bmr (or related metabolic rates) and the masses of energetically expensive internal organs. it is also important to note that measurements of bmr are technically complicated and inherently burdened with measurement error of 1520 % (konarzewski. it is very likely that the remaining variation can be attributed to fat and/or muscle mass. indeed, the inverse correlation between fat mass and bmr has been reported in many artificial selection studies (e.g. bunger. on the other hand, specific studies on the contribution of muscle mass to intra - specific variation in bmr in small mammals have not been carried out. interestingly, recent metabolomic analysis of metabolite profiles of mice selected for mass - corrected maximum metabolic rate suggest that bmr may increase due to elevated amino acid and energy metabolism in the musculature (wone. further such study would be very desirable, as at the inter - specific level, muscle mass, but not internal organ mass seems to explain most of the variation in bmr (raichlen. at rest, the mass - corrected metabolic rate of endotherms is 510 times higher than the mass - corrected metabolic rate of ectotherms (bennett and ruben 1979 ; hulbert and else 1981). at the cellular level, the major process that accounts for this difference is mitochondrial uncoupling and proton leak across the inner mitochondrial membrane, in mammals largely mediated by mitochondrial carrier proteins (porter. the most prominent of them is uncoupling protein-1 (ucp-1), which facilitates non - shivering thermogenesis in mammalian brown adipose tissue (bat ; cannon and nedergaard 2010). according to the membrane pacemaker theory of metabolism, the key factor behind the leakiness of the cell membranes is the chemical composition of their fatty acids, particularly the relative abundance of long - chain polyunsaturated fatty acids (pufas ; hulbert 2003 ; hulbert and else 1999, 2000, 2005). the chemical composition of fatty acids affects the physical properties of cell membranes, which in turn modulates the activity of many metabolically important enzymes and determines metabolic costs of maintenance of ionic gradients across cell membranes (else and wu 1999 ; wu. indeed, differences in fatty acyl composition of the mitochondrial membranes and in proton leak between ecto- and endotherms are well documented (e.g. mitchell. however, their contribution to inter - specific variation in bmr in mammals is less clear : proton leak does not explain differences in bmr between marsupials and eutherians (polymeropoulos. 2012), and there is no notable correlation between mammalian bmr and muscle phospholipid fatty acid composition (valencak and ruf 2007). direct evidence for the quantitative contribution of proton leak to within - species variation in bmr comes from two studies. rolfe and brown (1997) reported that as much as 20 % of bmr can be attributed to an incomplete coupling of substrate oxidation. it is therefore likely that a significant proportion of bmr variation is mediated through ucps. (2004) who found that individual mf1 mice having high bmr also have skeletal mitochondria characterized by high proton conductance. this increased conductance was caused by higher levels of endogenous activators of proton leak through the adenine nucleotide translocase and uncoupling protein-3 (ucp-3). on the other hand, however, mcdaneld. (2002) found that a response to selection for increased energy expenditure in mice selected for heat loss by nielsen. (1997a, b) was not mediated by increased expression or function of ucp-1 (for detailed characterization of this selection experiment see table 1). the mice in the low heat - loss line expressed significantly more ucp-1 mrna than did high heat - loss mice. (2002) also found that uncoupling protein-2 (ucp-2) mrna content was similar in mice characterized by high and low heat loss. thus, conspicuous differences in energy expenditure can be independent of ucp-1 and ucp-2-mediated thermogenesis. there is also no direct evidence for a correlation between cell membrane fatty acid composition and bmr within species as predicted by the membrane pacemaker theory. (2008) found no correlation between bmr and lipid desaturation in the liver of mf1 mice. they also did not find a correlation between bmr and either the proportion of oleic acid (18:1) or highly polyunsaturated 22:6 docosahexanoic (dha) fatty acid content in liver membranes. the lack of a correlation between bmr and the proportion of dha is particularly telling because dha has been identified as key component of the membrane pacemaker theory (hulbert and else 1999, 2000, 2005). (2007) analysed cell membrane fatty acyl composition in the liver and kidneys of mice divergently selected for bmr. contrary to the predictions derived from the membrane pacemaker theory the unsaturation index (the number of double bonds per 100 acyl chains) of the fatty acids in the kidney cell membranes did not differ between selected lines, despite 30 % difference in bmr. furthermore, the unsaturation index was higher in livers of mice from the low - bmr line, mainly because of a significantly higher content of dha. thus, divergent selection for bmr affected fatty acyl composition of phospholipids in the liver in the opposite direction to that predicted by the membrane pacemaker theory. it is important to note, however, that the lack of support for this theory does not necessarily question the contribution of the proton leak to bmr. k atpase for the maintenance of cell membrane ionic gradients has been implicated as another component of bmr, accounting for ca. 20 % of its variation (rolfe and brown 1997 ; wu. nevertheless, it is unclear how much of the costs of maintaining ionic gradients contribute to intra - specific variation in bmr. the cell metabolism hypothesis (kozowski. 2003) suggests an intriguing, yet untested, functional link between those costs and variation in bmr, mediated through variation in the size of individual cells constituting tissues and organs. it follows from the simple geometric relationship between surface area and volume that individuals of similar body mass, but built of larger cells should have relatively smaller cell summed surfaces than those, built of smaller, but more numerous cells. thus, all else being equal, a simple way to decrease / increase bmr is to decrease / increase the cell size of metabolically expensive tissues. to our knowledge, there are no published studies that allow for a direct test of this hypothesis in homeotherms. (2011) tested it indirectly by comparing the mass - corrected standard metabolic rate (smr) and erythrocyte size (used as a proxy for cell size, for justification see kozowski. 2010) between diploid and triploid individuals of a small fish belonging to the cobitistaenia hybrid complex. recently, maciak also found a similar inverse relationship between bmr and erythrocyte size in lines of mice divergently selected for bmr, with h - bmr individuals having 10 % smaller erythrocytes than those of l - bmr line (maciak, unpublished phd thesis). it is important to note in this context that the ploidy level, and therefore cell size, of metabolically expensive organs, such as liver, can vary within mammalian species, including humans (duncan. these observations provide empirical support for cell size as an important determinant of variation in bmr at the intra - specific level, though its generality remains to be established (kozowski. besides proton leak and the maintenance of ionic gradients, the third most important component of cellular metabolism is the cost of biosynthesis, comprising ca. interestingly, all three components are regulated by a single signalling pathway the mammalian target of rapamycin (mtor), which is therefore likely to be the most important molecular mechanism underlying the within - species variation in bmr (fig. 1 ; for review see laplante and sabatini 2009 ; ramanathan and schreiber 2009 ; schieke. the mtor is a serine / threonine protein kinase coded by a highly conserved tor gene found within every eucaryote genome (wullschleger. mtor forms two distinct complexes : (1) mtorc1, which is inhibited by antibiotic rapamycin and contains the protein component called raptor (fig. 1), and (2) mtorc2 insensitive to rapamycin, and in which mtor is bound to another protein partner called rictor (wullschleger. the pivotal role of mtor in cell size and growth regulation is well documented (guertin. the main environmental cue affecting mtor activity is the availability of nutrients, mostly amino acids and glucose. in the absence of amino acids the mtor signalling is inhibited and protein synthesis is thereby down - regulated, which arrests cell growth. cell metabolism is mainly regulated by mtorc1 through amp - activated protein kinase (ampk), which is in turn activated in response to a high amp / atp ratio within cells. (2006), used jurkat t cell leukaemia clone e6 - 1 cells as a model and found a positive correlation between resting mitochondrial respiration of individual cells and the activity of their mtor raptor complexes. they also demonstrated that inhibition of mtorc1 by rapamycin administration leads to the reduction of mitochondrial membrane potential, oxygen consumption, and consequently atp production. according to schieke. (2006), mtor activity is also likely to determine the balance between generation of atp through mitochondrial and non - mitochondrial cascades.fig. the mtor raptor complex responds to nutrient availability by up- or down - regulating mitochondrial oxidation. it also controls cell growth, which in turn generates metabolic costs of biosynthesis directly, and indirectly affects the metabolic costs of maintenance of the membrane ionic gradients being the function of the cell sizefig. 40 % of phenotypic variance can be attributed to additive genetic effects (table 1 in white and kearney 2012). thus, it is likely that in most populations the frequency of alleles underlying bmr is somewhere between two extremes : (1) the loss of genetic variation due to genetic drift or purifying selection, (2) the fixation of alleles due to long - term directional selection. 45 % of the total bmr variation can be due to environmental effects and non - additive gene expression. this points to the need to examine bmr variation using functional genomics tools schematic representation of regulation of bmr variation by the mtor pathway. the mtor raptor complex responds to nutrient availability by up- or down - regulating mitochondrial oxidation. it also controls cell growth, which in turn generates metabolic costs of biosynthesis directly, and indirectly affects the metabolic costs of maintenance of the membrane ionic gradients being the function of the cell size schematic representation of phenotypic variation in bmr.. 40 % of phenotypic variance can be attributed to additive genetic effects (table 1 in white and kearney 2012). thus, it is likely that in most populations the frequency of alleles underlying bmr is somewhere between two extremes : (1) the loss of genetic variation due to genetic drift or purifying selection, (2) the fixation of alleles due to long - term directional selection. assuming a 15 % measurement error of bmr (konarzewski. 45 % of the total bmr variation can be due to environmental effects and non - additive gene expression. high levels of bmr underlying endothermy have most likely evolved as a correlated response to selection for high rates of aerobic metabolism (bennett and ruben 1979) or an increased parental investment capacity (koteja 2000). theoretical analyses of the genetics of the evolution of endothermy provide opposing predictions with regard to determination of bmr and its covariation with other physiological traits. according to a strong version of the aerobic capacity model a positive genetic correlation between bmr and other traits (chiefly maximum metabolic rate, mmr) is an inexorable feature of the design of homeotherms, and therefore persisted not only at the early stages of bmr evolution, but is also present in extant birds and mammals (hayes 2010 ; nespolo., however, the weak form the aerobic capacity model predicts that directional selection was likely to result in the fixation of genes conferring a phenotypic advantage and, consequently, has resulted in a considerably reduced genetic variation of bmr and its covariation between traits (nespolo. 2011). according to this evolutionary scenario, genetic variation and covariation of bmr that was present in proto - endotherms may no longer be detectable in some or all extant lineages of homeotherms by means of classic methods of quantitative genetics. despite fundamental significance of the question of the extent of genetic determination of bmr, to date the great majority of studies on variation in bmr have focused on its phenotypic variation and discussed its adaptive significance based solely on non - genetic data (e.g. mcnab 2002). it is important to note, however, that phenotypic variation per se does not allow for meaningful evolutionary inference (roff 2002). likewise, phenotypic correlations between studied traits do not necessarily reflect their potential to respond to selection in a concerted fashion, as the strength (and even sign !) of phenotypic and genetic correlations may differ (roff 2002). only recently integrative physiologists have become aware of the need to study heritable variation in physiological traits (nespolo. for this reason, the number of studies on bmr heritability is limited and primarily restricted to classical laboratory model rodents (dohm. 2001 ; konarzewski. 2005) or wild species bred under laboratory conditions (nespolo. 2003, 2005 ; sadowska. 2005 ; rnning. a key quantitative genetic parameter, informing the potential of a given trait to respond to selection and therefore, to evolve, is the narrow - sense heritability (h), which is the ratio of additive genetic variance to total phenotypic variance (falconer and mackay 1996). early laboratory studies indicated very low or even insignificant narrow - sense heritability of bmr in laboratory mice (lacy and lynch 1979 ; lynch and sulzbach 1984 ; dohm. 2001) and a wild rodent, the leaf - eared mouse phyllotis darwini (nespolo. 2003 ; bacigalupe. 2004). furthermore, the heritability of some traits closely related to bmr, such as body temperature, is also effectively zero (lacy and lynch 1979 ; lynch and sulzbach 1984 ; nespolo. more recent studies, however, have found a relatively high and significant narrow - sense heritability of bmr in laboratory mice (h = 0.38, konarzewski. 2005 ; h = 0.19, wone. 2009), bank voles myodes (clethrionomys) glareolus (h = 0.4, sadowska. 2005) zebra finches taeniopygia guttata (h = 0.25, rnning. 2007) and least weasels (mustela nivalis, zub. thus, it seems that at least in some animal populations there is substantial additive genetic variation in bmr at the level of ca. also, the presence of a significant genetic component of human bmr was supported by studies on families participating in phase 2 of the qubec family study (rice. 2006), which is likely related to polymorphisms in the leptin and leptin receptor genes (loos. 2006). despite the unquestionable advantages of laboratory conditions, these studies yield estimates obtained in an artificial environment, and on animals that are more inbred than individuals in natural populations. constant laboratory - controlled conditions are also likely to inflate heritability estimates, because the effect of environmental variation is lower than in natural populations (riska. on the other hand, the propensity of bmr to be influenced by environmental factors such as temperature, food availability and photoperiod (mcnab 2002) make field estimates of bmr h very difficult. studies examining the heritability of metabolic traits in wild populations are even more complicated by difficulties associated with constructing a pedigree, which is necessary for calculating h (lynch and walsh 1998 ; coltman 2005). to date there are just two published studies estimating the h of bmr in the wild. (2009) found a significant h of bmr in a small, cavity - nesting passerine the blue tit (cyanistescaeruleus). this study used split cross - fostering of nestlings, which allowed for an effective separation of genetic and environmental effects on bmr for individuals in a known pedigree. unfortunately, for most wild populations information on environmental factors affecting individuals and their relatedness is unavailable. pedigree reconstruction, however, has now become possible thanks to the application of methods utilizing inference derived from the analysis of highly polymorphic molecular markers (for review see garant and kruuk 2005 ; pemberton 2008). thus, the reconstructed structure of relatedness can be combined with individual bmr measurements and analysed using a class of statistical analysis referred to as the animal model (for review see kruuk 2004 ; shaw 1987 ; thompson 2008). the animal model is based on restricted maximum likelihood (reml) computational techniques and consists of a mixture of both fixed and random effects, which allows for the effective partitioning of the phenotypic and genetic components of variance (wilson. 2009). (2012) used a marker - based approach to reconstruct the pedigree and then used an animal model to estimate the h of body mass and bmr in the free - living population of weasels mustela nivalis a small carnivore characterized by a wide range of body mass and extremely high bmr. (2012) found that the h of whole - body bmr and bmr was equal to 0.54 and 0.45, respectively, which are values comparable to laboratory h estimates. the study of zub. (2012) demonstrates clearly that marker - based approaches to pedigree reconstruction make it possible to analyse data for metabolic traits in wild populations. such data would otherwise be impossible to obtain in the absence of pedigree information.. this has become exceptionally important in the context of microevolutionary responses to climate change and the paucity of data for disentangling the genetic (evolutionary) and phenotypic (plastic) components of physiological mechanisms underlying those responses (for review see gienapp. the results of quantitative genetic analyses strongly suggest that a significant part of the phenotypic variance in bmr can be attributed to an additive genetic component, mostly underlined by many genes of small effect, coding for structural polymorphism of proteins. 40 % of bmr variation can be attributed to environmental effects and non - additive genetic effects, presumably acting through the modulation of gene expression (fig. 2 ; nespolo. 2011 ; for a concise review of the concepts see whitehead and crawford 2006). the relative contribution of genes underlying structural polymorphism and gene expression to the overall genetic variation of bmr remains to be determined. however, there is a mounting body of evidence that many phenotypic differences within and between populations are due to differences in gene expression (e.g. oleksiak. this might be the case for bmr, if underlying metabolic pathways were conserved in the course of its evolution and are mainly determined by the degree of expression of genes shared by ecto- and endotherms (seebacher. although this still remains to be tested, the advent of a new generation of dna sequencing and gene expression technologies brings the promise of a rapid progress in understanding the genetic / genomic basis of complex physiological traits, such as bmr (e.g. vera. nevertheless, there is already a wealth of information available on the genomics of traits that likely contribute to bmr, such as the mapping of qtls underlying aerobic capacity (moody. 2006) and the examination of gene expression in metabolically expensive tissues (klaus. the scope of this paper prevents us from a detailed appraisal of the genomics of metabolic rates, which certainly deserves a dedicated review. we have therefore decided to concentrate on the skeletal and heart muscles because many studies suggest that they significantly contribute to bmr (konarzewski and diamond 1995 ; raichlen. 2010), and focus on recent insights gleaned from artificial selection experiments and transgenic manipulations. one of the best studied models are mice (garland 2003 ; middleton. 2008) and rats (koch and britton 2001 ; wislff. koch and britton s experiment rats were divergently selected for exercise capacity by treadmill running at 11 weeks of age. given that the line selected towards high endurance running capacity was characterized by increased : (1) food consumption, (2) percent lean mass and (3) mass of metabolically active visceral organs (swallow. 2010), it seems reasonable to assume that the selection also resulted in a between - line divergence in bmr. however, despite the 120 % between - line difference in the primary selected trait, bye. (2008a) found only three differences in the expression of transcripts in the soleus muscle of rats of both lines, with unclear, immediate connection to bmr. much more conspicuous genetic differences have been found among lines of laboratory mice selected for increased levels of voluntary wheel running (middleton. mice from two of the four selected replicate lines exhibited dramatically increased locomotor activity and maximal oxygen consumption, as well as increased mass - specific muscle cellular aerobic capacity, heart size, and hindlimb bone lengths (for review see middleton. these effects were due to a mendelian recessive allele that, when present in the homozygous condition, caused a 50 % reduction in hindlimb muscle mass (garland. this gene has been mapped to a 2.6335-mb interval on the mmu11 region of chromosome 11, which harbours ca. 100 genes that are likely to underlie muscle development and function (hartmann.. it must be noted, however, that despite threefold differences in voluntary wheel running, the selected and control lines do not differ with respect to bmr (kane. 2008), which cautions against the existence of inescapable genetic link between bmr and aerobic capacity of skeletal muscles. this conclusion is corroborated by the lack of an effect of over - expression of mitochondrial uncoupling protein-1 (ucp-1) in skeletal muscles on bmr of hsa - mucp-1 transgenic mice (klaus. 2005). interestingly, compared with littermate controls, hsa - mucp-1 transgenic mice have substantially reduced levels of adiposity and increased total energy expenditures below the thermoneutral zone, most likely due to decreased muscle energy efficiency (klaus. 2005). the lack of an appreciable effect of transgenic manipulation of ucp-1on bmr is puzzling and clearly deserves further study. in contrast to small genetic differences underlying skeletal muscle function, koch and britton s (2001) selection experiment resulted in a considerable between - line difference in gene expression of the heart muscle (bye. interestingly, rats of hcr and lcr lines expressed genes underlying lipid and glucose metabolism, respectively. this suggests that the selection regime led to divergence in cardiac energy substrate utilization, towards mitochondrial fatty acid oxidation in hcr rats and glucose - based metabolism in lcr rats. bye. (2008b) linked those differences in expression to genes coding uncoupling protein-4 (ucp-4) in the hcr line, which is likely to be involved in the regulation of fatty acid -oxidation and therefore, influencing bmr. the existence of an association between the effects of artificial selection on aerobic capacity and the genetics of heart muscle metabolism has also been confirmed by babik. (2010) in a non - model rodent the bank vole (myodes glareolus). (2010) used 454 sequencing technology (for review see wheat 2010) followed by expression profiling of the heart transcriptome in lines of bank voles selected for high metabolism as compared to unselected controls (sadowska. they detected a number of putative single nucleotide polymorphisms (snps) between selection lines whose variant frequency differences were much higher than those expected by chance. although the exact causal link between identified snps and the underlying response to selection on metabolic rate remains unclear, babik.s (2010) study exemplifies the potential offered by new generation sequencing technologies for studying bmr in animals whose genome sequences are not available (see also vera. our review shows that intra - specific variation in bmr remains a viable source of information regarding metabolic expenditure, with clear functional links to key metabolic processes at all levels of biological organization. we also expose a number of unanswered questions and emerging research areas, which we summarize below. we have only briefly touched upon the discrepancies between the conclusions drawn from intra- and inter - specific studies on the significant factors affecting variation in bmr, such as the contribution of skeletal muscles (raichlen. 2010) and fatty acid composition of the cell membranes (polymeropoulos. 2012). although the directionality of the correlations between bmr and those components do not need to be the same at the inter- and intra - specific levels, the lack of congruency is puzzling in the context of the proposed mechanisms of the evolution of endothermy (nespolo. 2011). most likely, this inconsistency can only be resolved by additional, within - species studies on animals from yet untapped parts of the inter - specific spectrum used in establishing patterns reported by hulbert and else (2005), mitchell. an accumulating body of information suggests that bmr is a heritable trait, at least under laboratory conditions. it would be therefore instructive to move quantitative genetics analyses of bmr into the field. with the advent of modern molecular genetic techniques, reconstruction of pedigrees for otherwise elusive species no longer poses an insurmountable difficulty, as evidenced by an increasing number of field studies on the traits such as fur coloration and flight metabolism (e.g. nachman.. such integration of molecular genetics with a conventional metabolic field studies would greatly strengthen the inference on adaptive significance of metabolic variation, so far based primarily on phenotypic data (for review see whitehead 2012). likewise, the incorporation of functional genomics tools into studies on metabolic variation in the field is badly needed (rokas and abbot 2009). borrowing from already well advanced biomedical research on gene expression, functional genomics should greatly advance the connections between metabolic phenotype, genotype and fitness in natural populations. as an initial blueprint, students of bmr functional genomics can follow already successful studies on morphological traits (e.g. fur coloration) as well as genomic studies on metabolic traits in insects (wheat. another, promising approach to identifying metabolic underpinnings of bmr is offered by metabolomics (wone. both targeted and untargeted global metabolic profiling of tissues and organs (goodacre. 2004) can be used to generate and test the hypotheses regarding the physiological function underlying variation in bmr. our review shows that despite decades of research, the sources of intra - specific variation in bmr at organ, tissue and molecular levels are still not firmly identified. paradoxically, in this regard, studies on free - ranging animals can be greatly illuminated by medical research that is quickly advancing thanks to the application of imaging technologies combined with genomics and other omics research. while researching the literature for this review, we were struck by the poor exchange of information and ideas between researchers working on the physiology of metabolic rates within animals and humans. we propose that connecting the dots between metabolic studies on the whole - body level with mtor activity holds promise for a grand picture integrating the regulation of the key metabolic mitochondrial oxidation, maintenance of membrane ionic gradients and biosynthetic costs, which together are manifested as energy expenditures quantified as bmr (fig	basal metabolic rate (bmr) provides a widely accepted benchmark of metabolic expenditure for endotherms under laboratory and natural conditions. while most studies examining bmr have concentrated on inter - specific variation, relatively less attention has been paid to the determinants of within - species variation. even fewer studies have analysed the determinants of within - species bmr variation corrected for the strong influence of body mass by appropriate means (e.g. ancova). here, we review recent advancements in studies on the quantitative genetics of bmr and organ mass variation, along with their molecular genetics. next, we decompose bmr variation at the organ, tissue and molecular level. we conclude that within - species variation in bmr and its components have a clear genetic signature, and are functionally linked to key metabolic process at all levels of biological organization. we highlight the need to integrate molecular genetics with conventional metabolic field studies to reveal the adaptive significance of metabolic variation. since comparing gene expressions inter - specifically is problematic, within - species studies are more likely to inform us about the genetic underpinnings of bmr. we also urge for better integration of animal and medical research on bmr ; the latter is quickly advancing thanks to the application of imaging technologies and omics studies. we also suggest that much insight on the biochemical and molecular underpinnings of bmr variation can be gained from integrating studies on the mammalian target of rapamycin (mtor), which appears to be the major regulatory pathway influencing the key molecular components of bmr.
behcet 's disease is a systemic vasculitis characterized by recurrent oral and genital ulcers and ocular inflammation, and may involve the joint, skin, central nervous system and gastrointestinal tract. there is no specific test for behcet 's disease, and the diagnosis is based upon clinical criteria. although multiple diagnostic criteria have been established, there is no universally accepted definition of behcet 's disease. the prevalence of intestinal behcet 's disease has been variable in several reports and accounts for about 1 - 2% of behcet 's disease (1, 2). the clinical symptoms are wide and include anorexia, vomiting, dyspepsia, diarrhea, and abdominal pain. the typical endoscopic findings are segmental mucosal inflammation, punch - out, fissuring or aphthoid ulcers in the ileocecal area (3, 4). although strictures are unusual, transmural inflammation and fistulae are frequently observed (5). these findings are similar to those of crohn 's disease. however, longitudinal ulcers, cobblestone appearance, and granuloma formation are very rare findings in intestinal behcet 's disease. we experienced a rare case of systemic behcet 's disease with intestinal involvement similar to crohn 's colitis. a 39-yr - old female presented with recurrent oro - genital ulcers, erythema nodosum, and arthralgia. two years later, she was admitted to our hospital because of oral and genital ulcer, lower abdominal pain, and frequent diarrhea for 15 days. she had suffered from intermittent hematochezia and cramping abdominal pain for the previous two years. further history taking revealed she had a post - traumatic epilepsy by traffic accident 13 yr before and had taken anti - epilepsy drugs, carbamazepine and sodium valproate. on admission, her blood pressure was 90/60 mmhg, her pulse rate was 70/min, body temperature was 36.5, and respiration rate was 20/min. her abdominal pain was located in the bilateral lower quadrants without rebound tenderness. she appeared chronically ill and reported that her stools were maroon, followed by mucoid. on examination of external genitalia, a linear to ovoid shaped laboratory values showed a white blood cell count of 8,600/l, hemoglobin of 9.1 g / dl, and a platelet count of 172,000/l. her liver function test showed aspartate transaminase of 13 iu / l, alanine transaminase of 6 iu / l, albumin of 2.3 g / dl, alkaline phosphatase of 357 u / l, and total bilirubin of 0.2 mg / dl. the other laboratory findings showed fast blood sugar of 119 mg / dl, blood urea nitrogen of 5.2 mg / dl, creatinine of 0.8 mg / dl, sodium of 136 meq / l, and potassium of 3.3 meq / l. an esophagogastroduodenoscopy (egd) was performed to rule out the presence of a massively bleeding upper gastrointestinal lesion. colonoscopic examination revealed grossly normal - appearing mucosa in the rectum and the sigmoid colon. however, descending, transverse, and ascending colon, including the cecum, showed multiple longitudinal ulcers and inflammatory pseudopolyps with a cobblestone appearance. the ileocecal valve and terminal ileum were preserved from the inflammation. a suspicious fistular opening just above the anus microscopically, specimens from ulcers of the colon showed shallow ulcerations with inflammatory infiltration consisting of lymphocytes and plasma cells. under sterile condition, intradermal injection of the skin with a 20-guage needle was done. within 48 hr, histological examination of the perineal lesion revealed chronic ulcer with acute and chronic inflammatory cell infiltration and increased small blood vessels (fig. total parenteral nutrition with nothing per oral and intravenous administration of methylprednisolone, 62.5 mg were started. treatment with prednisone, 40 mg and 5-aminosalicylate (mesalazine), 4.0 g by mouth was started. the abdominal symptoms and vulva ulcer improved gradually, and she was discharged 2 months after admission. behcet 's disease was originally described in 1937 and characterized by oral and genital ulceration and ocular inflammation (6). the diagnosis is clinical and is now based on criteria suggested by an international study group for behcet 's disease (7). in the present case, oro - genital ulceration and skin lesions led to the diagnosis of behcet 's disease. the etiology remains unknown, but the most widely held hypothesis of pathogenesis is that a profound inflammatory response is triggered by an infectious agent in a genetically susceptible host (6). other possible mechanism is that behcet 's disease may be autoimmune in origin (8, 9). an inflammatory response to several autoantigens is found, and generalized aberrant t cell responses results in enhanced nonspecific inflammation. an increased production of interferon gamma (ifn-) by t cells has been demonstrated in active behcet 's disease, and circulating t cells have the t - helper phenotype (th1) predominantly (10). in crohn 's disease, murine and human studies have demonstrated an increased expression of th1 cytokines by lamina propria lymphocytes (11). oshima. (12) reported that over 40% of behcet 's disease patients had gastrointestinal complaints. this so - called intestinal behcet 's disease accounts for only 1 - 2% of cases (1, 2). in intestinal behcet 's disease, ulceration of the gastrointestinal tract can be found throughout the intestine, but the most frequent area of involvement is the ileocecal region. behcet 's colitis can appear as ulcerative colitis or crohn 's disease when there are skip lesions with rectal sparing. the ulcers are usually large, discrete, punch - out appearing lesions and can extend to the serosa. formation of fistula, hemorrhage, or perforation occurs in up to 50% of cases involving the intestine. the lymphocytes are infiltrated, and dense perivascular infiltrate is frequently seen (3, 4). there are many extra - intestinal findings of crohn 's disease, such as oral and genital ulcers, erythema nodosum, uveitis and arthritis, resembling the manifestations of behcet 's disease. it is also very difficult to distinguish the intestinal behcet 's disease from that of crohn 's disease in some patients. it is possible that a patient with crohn 's disease meets the criteria for behcet 's disease. there are several reports on the coexistence of behcet 's disease and crohn 's disease (14, 15). in the present case, we made the diagnosis of behcet 's disease as described above. however, the gastrointestinal manifestation of this case is quite atypical for behcet 's colitis and more similar to crohn 's colitis - longitudinal ulcers, cobblestone appearance, and ano - rectal fistula are usual findings in crohn 's colitis. however, we can not conclude that the patient had crohn 's disease because granulomas, the hallmark of crohn 's colitis, were absent. the findings in the family reported by yim and white (16), suggest that inflammatory bowel disease and behcet 's disease may be closely related and part of a spectrum of disease rather than distinct disease entities. in our patient, patient 's bowel symptoms, endoscopic appearance, and the response to medical treatment were compatible with crohn 's colitis. these findings suggest that behcet 's disease may be a part of the spectrum of chronic inflammatory bowel disease.	behcet 's disease is a multi - systemic vasculitis and characterized by systemic organ involvement. although the gastrointestinal and systemic features of behcet 's disease and inflammatory bowel disease overlap to a considerable extent, they are generally viewed as two distinct diseases. a 39-yr - old female was diagnosed as having behcet 's disease. she was admitted to our hospital because of oral and genital ulcer, lower abdominal pain, and frequent diarrhea. colonosopy showed diffuse involvement of multiple longitudinal ulcers with inflammatory pseudopolyps with a cobblestone appearance and ano - rectal fistula was suspected. these findings are extremely rare in behcet 's disease. however, there were no granulomas, the hallmark of crohn 's colitis. microscopically, perivasculitis and multiple lymph follicles compatible with behcet 's disease were seen. although being rarely encountered, multiple longitudinal ulcers, cobblestone appearance, and ano - rectal fistula can develop in behcet 's disease, as in crohn 's colitis. therefore, behcet 's disease and crohn 's disease may be closely related and part of a spectrum of disease.
global incidence of diabetes mellitus (dm) estimates more than 171 million for 2000 and 366 million for 2030. the mortality and morbidity of dm are determined by various complications, such as diabetic vasculopathy, retinopathy, nephropathy, and peripheral neuropathy. recently, many studies have indicated that dm also implicated the central nervous system (cns) and induced the brain pathological changes, named the diabetic encephalopathy, which is a complication of dm in the cns characterized by mild cognitive deficits and neuropathology [35 ]. diabetic encephalopathy presents many symptoms, which can be described as the features of brain aging including brain atrophy, reactive oxygen species (ros) accumulation, cerebral vasculopathy, and impairment of cognition [6, 7 ]. clinical observation has shown that brain atrophy is more remarkable in diabetic patients than in age - matched controls. animal experimental data have suggested that learning deficiency is associated with the distinct changes in synaptic plasticity in hippocampal slices in streptozotocin- (stz-) induced diabetic rats. the affinity of glutamate for ampa but not for nmda receptors decreases in sprague - dawley (sd) rats at 6 to 8 weeks after stz injection. the levels of malondialdehyde, xanthine oxidase, and nitric oxide in the hippocampus, cortex, cerebellum, brain stem, and spinal cord significantly increase in stz - induced diabetic - untreated rats, suggesting remarkable generation of ros in the brain. hyperglycemia resulting from defective insulin secretion, resistance to insulin action, or both is a critical pathogenesis of dm. willem hendrik gispen and geert - jan biessels have recently suggested that acute hyperglycemia is associated with mild cognitive dysfunction in population with type 1 or type 2 dm. another recent study has suggested that insulin is implicated in the pathogenesis of age - related memory decline and diabetic encephalopathy [13, 14 ]. insulin may act as a neuromodulator that regulates the release and reuptake of neurotransmitters and probably affects learning and memory. impairments in the insulin signaling pathway in the periphery and brain have been implicated in alzheimer 's disease, diabetes, and aging [16, 17 ]. recent studies have revealed that impairments in cerebral glucose utilization and energy metabolism represent early abnormalities that precede or accompany the initial stages of cognitive impairment. cerebral glucose utilization deficiency and insulin signaling decline are common features between dm and alzheimer 's disease (ad). ad is a progressive neurodegenerative disease characterized by the loss of memory and other cognitive functions, resulting in dementia. the hallmarks of pathology of ad are a deposition and microtubule - associated protein tau overphosphorylation and formed the senile plaques in the extracellular matrix. additionally, some studies have indicated that the insulin signals are involved in regulation of a accumulation and tau phosphorylation [17, 21 ]. furthermore, the factors associated with high risk of ad are also involved in the development of dm, especially t2 dm. clinical and experimental data clearly showed that diabetes affected the brain and these effects are similar to the acceleration of brain ageing. however, whether or not diabetic encephalopathy at early onset shows ad - like pathology as aging dependent neurodegenerative disease remains unclear. in the present study, changes in the brain were indicated by hippocampal atrophy accompanied with beta - amyloid deposition, synapse loss, and learning behavioral deficiency at 4 months after stz injection. fiftymale sprague - dawley (sd) rats (810 weeks old) were obtained from the experimental animal center of lanzhou university. the rats were kept in an animal house at 22 2c temperature, 65 10% relative humidity, and 12 h light / dark cycle. fluoro - jade c (fjc) and mouse monoclonal anti - gapdh and rabbit polyclonal anti - a42 antibodies were purchased from millipore (bellerica, ma, usa). glucose, total cholesterol, triglyceride, and creatine enzymatic diagnostic kits were purchased from randox (crumlin, county antrim, uk). rabbit polyclonal anti - synaptophysin (syn) antibody was purchased from santa cruz biotechnology incorporation (santa cruz, ca, usa). thirty rats were fasted overnight and then injected with stz (65 mg / kg body wt.) through the femoral vein. one week after stz injection, blood samples were collected through the tail vein, and plasma glucose level and insulin level were measured by plasma glucose enzymatic diagnostic kits and insulin elisa kit, respectively. rats with the plasma glucose level 300 mg / dl and symptoms of polyuria, polyphagia, and polydipsia were considered to be diabetic and used in the present study. the fasting plasma insulin level in stz - induced dm rats was 1.32 0.6 pmol / l and was markedly lower than that of the normal rats (166.2 4.3 the metabolic parameters were tested according to our previous methods. in brief, the rats were placed in metabolic cages and raised for 24 h after 8 h of fasting. urine/24 h, water/24 h, and food consumption/24 h were measured, and fasting plasma glucose, triglyceride, total cholesterol, and creatinine were quantified using respective enzymatic diagnostic kits. creatinine clear ratio (ccr) was calculated according to urine volume/24 h and body weight ; the ccr was calculated as follows : (1)urine creatinineurine volume (ml/24 h)(plasma finally, the total metabolic data of 18 rats in each group were recorded after four months. six rats in each group were sacrificed with an overdose of 10% chloral hydrate and then transcardially perfused with 0.9% saline solution followed by 4% ice - cold phosphate - buffered paraformaldehyde (pfa). the brains were removed, postfixed in 4% pfa for 12 h, and then immersed sequentially in 20% and 30% sucrose solutions in 0.1 m phosphate buffer (ph 7.4) until they sank. coronal sections with a thickness of 40 m were cut at 2.2 mm to 4.80 mm from the bregma using a freezing microtome (leica, germany) and then stored at 20c in a cryoprotectant solution. to evaluate brain atrophy, the forebrain and hippocampal volumes were measured according to our previous methods. in brief, for forebrain volume, six sections at 2.28 mm to 0.12 mm from the bregma (interval at 400 m) were selected and then stained with cresyl violet. the forebrain volume was calculated as (2)[s1a+s2a2+s2a+s3a2+s3a+s4a2 + s4a+s5a2+s5a+s6a2]400, where s1a to s6a represent the forebrain areas in sections 1 to 6, respectively. for hippocampal volume, six sections at 2.2 mm to 4.6 mm from the bregma (interval at 400 m) were selected and then stained with cresyl violet. the hippocampal volume was calculated as (3)[s1a+s2a2+s2a+s3a2+s3a+s4a2 + s4a+s5a2+s5a+s6a2]400, where s1a to s6a represent the hippocampal areas in sections 1 to 6, respectively. to evaluate the neurodegeneration, the fjc staining was used according to our previous methods. in brief, six sections at 2.28 mm to 0.12 mm and six sections at 2.2 mm to 4.6 mm from the bregma (at 400 m intervals) were selected, respectively. dried sections were dipped in 80% ethanol solution that contains 1% sodium hydroxide, 70% ethanol, and 0.06% potassium permanganate for 5, 2, and 10 min, respectively. the sections were rinsed with distilled water and then incubated with 0.0004% fjc in 0.1% acetic acid for 20 min. fjc staining was detected under a fluorescent microscope at 480 nm excitation and 525 nm emission. images were acquired through a 20 objective, and the number of fjc - positive cells in the frontal cortex, hippocampus, and hypothalamus was counted. the total number of positive cells in the frontal cortex was calculated as (4)[s1+s22 + s2+s32+s3+s42+s4+s52+s5+s62]10, where s1 to s6 represent the fjc - positive cell numbers in sections 1 to 6, respectively. the total number of positive cells in the hippocampus and hypothalamus was calculated as (5)[s7+s82+s8+s92+s9+s102 + s10+s112+s11+s122]10, where s7 to s12 represent the fjc - positive cell numbers in hippocampus or hypothalamus in sections 7 to 12, respectively. for a42 immunostaining, three sections at 2.28 mm, 0.12 mm, and 2.2 mm from the bregma were selected and then incubated with 0.3% h2o2 for 30 min. the sections were placed in blocking buffer that contains 10% normal goat serum and 0.3% triton x-100 in 0.01 m phosphate - buffered saline (ph 7.2) for 30 min at 37c and then incubated overnight with antibodies against rabbit polyclonal anti - a42 (1 : 500) at 4c. the sections were then incubated with corresponding biotinylated secondary antibodies (1 : 200) at 37c for 1 h and then with avidin - biotin - peroxidase (1 : 200) at 37c for 1 h. immunoreactivity was visualized with 0.05% 3, 3-diaminobenzidine as chromogen. negative controls received the same treatment without the primary antibodies and showed no specific staining. six rats were sacrificed by decapitation, and their brains were quickly removed and placed on ice - cold glass plates. the hippocampus was rapidly dissected, frozen, and then stored in a deep freezer at 80c until assayed. the frozen tissues were homogenized in 4 volumes of buffer a that contains 50 mm tris - hcl (ph 7.6), 150 mm nacl, and a protease inhibitor cocktail (complete ; roche diagnostics, mannheim, germany) with 10 strokes of a teflon glass homogenizer. the supernatant was used as the soluble fraction, and the pellet was solubilized by sonication in buffer a that contains 6 m guanidine - hcl. the solubilized pellet was centrifuged at 20,000 g for 20 min at 4c. the supernatant was diluted 12-fold to reduce the concentration of guanidine - hcl and used as the insoluble fraction. golgi staining was performed on 4-month - old diabetic rats and age - matched normal rats according to the previous methods. briefly, freshly dissected brains were immersed in a golgi - cox solution for 2 weeks at room temperature. the golgi - cox solution was replaced, and immersion was continued for 2 weeks. the pyramidal neurons in cortical layers ii / iii and in the ca1 region of the hippocampus were analyzed. a minimum of two segments were randomly selected per neuron from the apical oblique and basal shaft dendrites. fourrats in each group were sacrificed by decapitation, and their brains were quickly removed and placed on ice - cold glass plates. the cerebral cortex was dissected and frozen in liquid nitrogen. total rna in the left cerebral cortex was extracted by trizol reagent, and rna concentration was measured using a spectrofluorometer. total protein in the right cerebral cortex was extracted by ripa buffer, and protein concentration was measured using the bradford assay. the frontal cortex and hippocampus of the rats were placed on ice - cold glass plates. proteins (50 g) were fractionated on 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis and then transferred into polyvinylidene fluoride membranes. the membranes were blotted with anti - synaptophysin (1 : 1000) and anti - gapdh (1 : 5000) antibodies, as well as with horseradish peroxidase - conjugated second antibody (1 : 5000). morris water maze was performed as previously described. briefly, at 4 months after stz injection, twelve rats were selected according to the open field test and then allowed to perform a learning task in the morris water maze with the blind method. the maze consisted of a black pool (148 cm diameter) filled with water (26 2c). a circular black platform was submerged 2 cm below the water surface in the middle of the target quadrant. the behavior of the rats in the pool was traced with a camera connected to a wmt-100 analysis system (taimeng, chinese instruments). the swimming speed, distance, and latency of the rats to find the platform were measured. the pool was divided into four quadrants (i.e., quadrants 1 to 4). the quadrants had different shapes, different colors, and three rings (inner, middle, and outer). the platform was placed in a constant location in the middle ring of quadrant 3. each day involved training the rats in the four quadrants with 30 min intervals. each trial was started by placing a rat with its back facing toward the platform at the starting points. when the rat did not find the platform within 60 s, it was guided on the platform for 15 s. during acquisition (days 1 to 4) of the spatial navigation test, all groups were given one session of four trials each day. on each day after training, the rat was removed from the pool, dried, and then returned to its cage. briefly, the ia box consisted of a lighted (safe) compartment and a dark (shock) compartment separated by a door. in the dark compartment, the rats received a footshock of 0.6 ma for 2 s through a constant current scrambler circuit delivered through the grid floor. rats were placed in the lighted compartment, as specified in each experiment, facing away from the door. the door leading to the dark chamber was opened after 10 s. once the animals fully entered the dark chamber (all four limbs contacting the grid in the dark chamber), the door was closed, and a footshock was delivered for 2 s after a 2 s delay. the rats were returned to their home cage 10 s later and were tested 48 h after training. mann - whitney u test was used for each evaluation, and unpaired student 's t - test was used for other data. the results of consecutive metabolic test showed that the body weight (figure 1(a)) of the diabetic rats was reduced compared with that of the age - matched rats. this test also demonstrated that the water consumption (figure 1(b)), food consumption (figure 1(c)), and urine production (figure 1(d)) of the diabetic rats increased compared with those of the age - matched rats. consecutive plasma examination showed that the levels of glucose (figure 1(e)), triglyceride (figure 1(f)), and cholesterol (figure 1(g)) were higher in the diabetic rats than in the age - matched rats. moreover, ccr was higher in the diabetic rats than in the age - matched control rats ; this result was more pronounced at 2 months after stz injection, suggesting that the kidney function was normal and that muscle wasting increased in the diabetic rats (figure 1(h)). to evaluate the brain results showedthe forebrain volume is not significantly different in the diabetic rats compared to the age - matched control rats at 2 and 4 months after stz injection (figures 2(a) and 2(b)). the hippocampal volume in the diabetic rats decreased at 4 months after stz injection compared with that in the age - matched control rats (figures 2(c) and 2(d)). the concentrations of rna and protein are shown in figures 2(e) and 2(f). the total rna concentrationin the cerebral cortex of the diabetic rats decreased at 4 months after stz injection (3.032 1.05 g/l) compared with that in the cerebral cortex of the age - matched control rats (4.56 1.21 g/l). the total protein concentration in the cerebral cortex of the diabetic rats also decreased at 4 months after stz injection (16.535 2.76 g/l) compared with that in the cerebral cortex of the age - matched control rats (19.805 2.49 g/l). the effect of the persistent metabolic disorder on neuronal survival was determined by conducting fjc staining to evaluate the neurodegeneration in discrete brain regions. the number of fjc - positive cells was higher in the frontal cortex (figures 3(a) and 3(b), 1570 350), hypothalamus (figures 3(c) and 3(d), 2800 385), and hippocampus (figures 3(e) and 3(f), 3235 502) of the diabetic rats at 4 months after stz injection than in those of the age - matched control rats. a deposition is the hallmark characteristic in ad and ad - like brain aging ; in the present study, a deposition was analysis. immunohistochemistry revealed the presence of a42 immunoreactivity in the frontal cortex (figure 4(a)) and hippocampus (figure 4(b)) of the diabetic rats at 4 months after stz injection. quantitative analysis of a42 in the frontal cortex and hippocampal tissues by elisa showed no significant difference in the soluble fraction of hippocampal tissues between the diabetic rats and age - matched control rats. however, the amount of a42 in the insoluble fraction increased in the diabetic rats at 4 months after stz injection compared with that in the age - matched control rats (figures 4(c) and 4(d)). to evaluate the synaptic plasticity, dendritic spine density and syn expression were measured. results showed that the dendritic spine density of the frontal cortex decreased in the diabetic rats at 4 months after stz injection compared with that in the age - matched control rats (figures 5(a) and 5(c)). consistently, the synaptophysin expression of the frontal cortex was lower in the diabetic rats than in the control rats (figures 5(d) and 5(f)). similarly, the dendritic spine density of the hippocampus decreased in the diabetic rats at 4 months after stz injection compared with that in the age - matched rats (figures 5(b) and 5(c)). the synaptophysin level in the hippocampal tissues decreased in the diabetic rats compared with that in the age - matched control rats (figures 5(e) and 5(f)). at 1 d after training, no significant difference in the time to find the platform (escape latency) was found between the diabetic rats and age - matched control rats at 4 months after stz injection. at 2, 3, and 4 d after training, the escape latency exhibited by the diabetic rats was significantly longer than that exhibited by the control rats (figures 6(a) and 6(b)). the velocity of swimming has no significant difference in both groups (figure 6(c)). no significant difference in training of ia was observed between the diabetic rats and age - matched control rats at 4 months after stz injection. however, at 24, 48, and 72 h after training, the freezing duration exhibited by the diabetic rats was notably shorter than that exhibited by the control rats (figure 6(d)). in this study, cognitive impairment accompanying hippocampal atrophy was observed at 4 months after stz injection. brain atrophy, as a general feature during brain aging, is involved in complicated mechanisms. stz - induced animal models are characterized by insulin deficiency accompanied with polydipsia, polyphagia, polyuria, and weight loss. insulin receptors are distributed in several brain regions, including the hippocampus, hypothalamus, olfactory bulb, and cortex. and plasma insulin can pass the blood - brain barrier, binding with its receptor to play important roles. in the cns, insulin is involved not only in regulating the glucose metabolism, but also in maintaining the neuronal survival and development. in the hippocampus the administration of insulin enhances performance in a passive - avoidance memory task, and spatial memory training alters the expression levels of insulin receptors in the hippocampus. consistently, recent animal studies have suggested that delivery of insulin to the hippocampus modulates hippocampal memory processes. therefore, insulin deficiency may play pilot roles in development of diabetic encephalopathy in stz - induced animal model. our results indicated that the dendritic spine density in the frontal cortex and hippocampus decreased in the stz - induced diabetic rats compared with the age - matched control rats. synaptophysin (syn), a crucial presynaptic protein, also decreased in the diabetic rats compared with age - matched control rats. aside from hyperglycemia due to insulin deficiency brain is the most cholesterol - rich organ in the body ; most of this cholesterol is produced in situ. the notion that decreased cholesterol biosynthesis alters the brain function is supported by the results of in vitro studies that cholesterol is essential for synaptogenesis and synapse function. in our present experiment, the insulin deficiency and aberrant lipid metabolism may contribute to the decline of synaptic plasticity. in the present study, a42 deposition in the hippocampus however, it did not form plaques in the extracellular matrix at 4 months after stz injection. at early onset, a aggregated in the cytoplasm. furthermore, a formed plaques and deposited in the extracellular matrix with the decline in a clearance. alternatively, the autopsy studies in humans do not find increased amyloid and tau pathology in relation to diabetes. but many studies indicated that the a aggregation was regulated by insulin signals in vivo and in vitro. once insulin has bound to its receptor, phosphoinositide 3-kinase is phosphorylated, and protein kinase b is activated, which in turn phosphorylates and thereby inhibits gsk-3. gsk-3 is a serine / threonine kinase encoded by two genes with high sequence homology (gsk-3 and gsk-3). also, insulin improves a clearance through insulin - degrading enzyme, one of the main proteases involved in a degradation. in the present study, the mechanisms of a deposition induced by dm remain to be elucidated in future work. in addition, our unpublished work indicated that the gsk3 activity increased in the hippocampus and frontal cortex of stz - induced diabetic rats. diabetes is characterized by marked peripheral alterations in glucose homeostasis, which leads to hyperglycemia. excess glucose is metabolized through alternative metabolic pathways, which may have direct adverse effects on the brain through the generation of potentially toxic metabolic by - products and through the depletion of important metabolic cofactors. in addition, diabetes is associated with long - term complications in other organ systems, wherein vascular disease can cause secondary damages to the brain. identifying the brain injury and cognitive impairment resulting only from central nervous systemic pathology is difficult because vascular pathology and metabolic toxicity may be important in diabetic encephalopathy development. after all, more and more evidence indicates that population with the type 1 or type 2 dm are prevalence in suffering from ad. and the rodent animal model with type 1 or type 2 dm can be observed the ad pathological changes including a deposition and tau - phosphorylation [40, 41 ]. in conclusion, aberrant metabolism including hyperglycemia and hyperlipidemia following insulin deficiency caused hippocampal atrophy, neurodegeneration, a deposition, and declined dendritic spine density in stz - induced diabetic rats.	objective. numerous epidemiological studies have linked diabetes mellitus (dm) with an increased risk of developing alzheimer 's disease (ad). however, whether or not diabetic encephalopathy shows ad - like pathology remains unclear. research design and methods. forebrain and hippocampal volumes were measured using stereology in serial coronal sections of the brain in streptozotocin- (stz-) induced rats. neurodegeneration in the frontal cortex, hypothalamus, and hippocampus was evaluated using fluoro - jade c (fjc). a aggregation in the frontal cortex and hippocampus was tested using immunohistochemistry and elisa. dendritic spine density in the frontal cortex and hippocampus was measured using golgi staining, and western blot was conducted to detect the levels of synaptophysin. cognitive ability was evaluated through the morris water maze and inhibitory avoidant box. results. rats are characterized by insulin deficiency accompanied with polydipsia, polyphagia, polyuria, and weight loss after stz injection. the number of fjc - positive cells significantly increased in discrete brain regions of the diabetic rats compared with the age - matched control rats. hippocampal atrophy, a aggregation, and synapse loss were observed in the diabetic rats compared with the control rats. the learning and memory of the diabetic rats decreased compared with those of the age - matched control rats. conclusions. our results suggested that aberrant metabolism induced brain aging as characterized by ad - like pathologies.
from november 26 to december 28, 2010, 6 cases of subtype h5n1 infection were identified in carcasses, feces, and cloacal swab specimens of migratory birds collected throughout south korea. subtype h5n1 viruses were found in various bird species (mallard, baikal teal, mandarin duck, whooper swan, and eurasian eagle owl) in places such as migratory bird habitats and nearby hills (figure 1, panel a). despite the repeated predictions from animal health authorities of poultry outbreaks and the emphasis on protection against contamination, on december 30, 2010, hpai was confirmed on 2 poultry farms. these farms are located in the middle region of south korea and are close (distances of 1.3 km and 0.4 km, respectively) to the migratory habitats of birds that have been positive for subtype h5n1 virus (figure 1, panel b). during the next 2 weeks, the percentage of hpai - positive birds increased rapidly among clustered poultry farms located in the southern region, and poultry on 23 farms and 13 wild birds were confirmed to be infected with hpai virus (figure 1, panel c). the hpai outbreak spread more slowly until may 16, 2011, and an additional 30 poultry farms and 7 wild birds were confirmed to be infected with subtype h5n1 virus (figure 1, panel d). progress of highly pathogenic avian influenza (hpai) outbreak by time, south korea, 20102011. a) hpai - positive cases identified from samples collected november 26december 28, 2010 (wild birds, 6 cases). b) cases identified by january 4, 2011 (wild birds, 10 cases ; poultry, 2 cases). c) cases identified by january 11, 2011 (wild birds, 13 cases ; poultry, 23 cases). d) cases identified by may 16, 2011 (wild birds, 20 cases ; poultry, 53 cases). blue circles indicate locations where hpai viruses were isolated from wild birds ; red circles indicate locations where hpai viruses were isolated from poultry. fourteen bird species were found to be positive for subtype h5n1 (table). the affected poultry included species of the order galliformes (chickens, quail, etc.), which exhibited sudden death with severe clinical signs, and domestic ducks (order anseriformes), which died suddenly or exhibited a decrease in egg production, depending on age. most infected birds of species that belonged to the orders anseriformes, falconiformes, and strigiformes were found dead, but a few infections were detected in swab specimens from healthy mallards and feces of wild birds. moreover, species of anseriformes, such as the mandarin duck, were dominant among the wild birds with hpai until the beginning of january 2011. after that time, many hpai infections were found in birds of prey, such as the eurasian eagle owl (table). all viruses were isolated by inoculating embryonated chicken eggs with specimens from cloacal swab specimens, feces, and homogenized organs from birds with suspected infections. the hemagglutinin (ha) and neuraminidase (na) proteins were subtyped as previously described (6). we selected 27 viruses, taking into consideration the outbreak period, the region, and the host species (table a1) and conducted sequencing and phylogenetic analysis of 8 gene segments. the genome sequences of 27 viruses are available from genbank under accession numbers jn807892jn808107. in the ha phylogenetic tree, all 27 viruses were clustered into clade 2.3.2 hpai viruses, together with the subtype h5n1 virus that had been isolated from a healthy mallard (7). all isolates showed a high ha homology (> 99.5%) (table a1). of note, the isolates from poultry fell into 2 sublineages, south - middle and north - middle, which were distinct geographic regions in the hpai outbreak among poultry, but the isolates from wild birds were not subgrouped in the phylogenetic tree, which displays only topology (figure 2). phylogenetic diagram of hemagglutinin (ha) gene of highly pathogenic avian influenza (h5n1) viruses, including viruses isolated in south korea during 20102011. blue indicates viruses isolated from wild birds, boldface indicates isolates from poultry, and red indicates reference virus. these isolates were different from the hpai viruses responsible for previous outbreaks in south korea (a / chicken / korea / es/2003[clade 2.5 ], a / chicken / korea / is/2006[clade 2.2 ] and a / chicken / korea / gimje/2008[clade 2.3.2 ]) and were closely related (> 99%) to the subtype h5n1 isolates found in mongolia, china, and russia in 20092010. the phylogenetic analysis also showed that the na and other internal genes were closely related to those of subtype h5n1 viruses found in wild birds in mongolia, china, and russia in 20092010 (figure a1 ; unpub. all 27 viruses characterized were highly pathogenic and had variations in the multibasic cleavage site in the ha molecule (pqrerrrkr) and a 20-aa deletion in the stalk region of na. they did not have amino acid substitutions that conferred resistance to amantadine or oseltamivir and were associated with the increased virulence of subtype h5n1 viruses in mammalian hosts (8). the intravenous pathogenicity test was conducted by using the a / duck / korea / cheonan/2010 virus, the first isolate from poultry. after the outbreak at lake qinghai, china, in 2005, the clade 2.2 viruses spread from asia to europe and africa during 20052006 and have been circulating widely in southern asia, the middle east, europe, and africa for several years. clade 2.3.2 viruses might spread over an extensive area, similar to clade 2.2 viruses, because clade 2.3.2 viruses are widespread among wild birds and have been continuously evolving in the regions where subtype h5n1 viruses are endemic (9). clade 2.3.2 viruses have circulated in vietnam and southern china since 2005, and clade 2.3.2 viruses that had undergone reassortment with clade 2.3.4 viruses were isolated from wild birds in hong kong in 2007. these reassorted viruses caused hpai (h5n1) outbreaks in japan, russia, and south korea during 2008 (6,1012). new 2.3.2 viruses, reassortants that possessed a different acidic polymerase gene from the 2.3.2 viruses of 20072008, were isolated predominantly from migratory birds in mongolia and china in 20092010 (13,14). no hpai outbreaks occurred in south korea and japan in 2009, but outbreaks of a similar virus took place in both countries in late 2010 (15). the situation was analogous to outbreaks in 2 countries in 20062007 by clade 2.2 hpai viruses that had been detected in china, mongolia, and russia in 2005. thus, the migratory patterns of infected wild birds might be related to these outbreaks. during the initial stage of the 20102011 outbreak, hpai viruses were detected in several wild birds, and the viruses were assumed to have been introduced into domestic poultry by migratory birds. the detection of hpai (h5n1) virus in free - ranging migratory bird might predict a poultry outbreak if biosecurity measures in poultry are inadequate, but during 2006, several european countries reported hpai (h5n1) virus infections in wild birds without concurrent poultry outbreaks. in the 2010 - 2011 outbreak in south korea, the subsequent outbreak cases suggest that the subtype h5n1 virus was spread from farm to farm by humans and associated agricultural practices so that strains of poultry were grouped in sublineages by region. clade 2.3.2 subtype h5n1 viruses have been circulating in poultry and migratory birds in asia and have accumulated antigenic mutations. we can conclude that early detection of hpai outbreaks and a rapid response to them are essential in controlling the introduction of virus from migratory birds to poultry and in preventing farm - to - farm spread.	highly pathogenic avian influenza (h5n1) among wild birds emerged simultaneously with outbreaks in domestic poultry in south korea during november 2010may 2011. phylogenetic analysis showed that these viruses belonged to clade 2.3.2, as did viruses found in mongolia, the people s republic of china, and russia in 2009 and 2010.
traffic injuries are a leading cause of death and disability in many countries, and they are major public health problems (14). it is anticipated that the traffic accident rate could increase by 50% by the end of 2020 if appropriate actions are not taken to reduce global deaths due to traffic accidents (1). some epidemiological studies have reported that the incidence rates of fatal traffic injuries are about 26.4, 17.4, and 19 per 100,000 people in the eastern mediterranean region, in the european region, and worldwide, respectively (5). in 2007, 27,567 deaths and 276,762 injuries as the result of traffic accidents were reported in iran (1). the knowledge, attitudes, and practice of drivers towards traffic regulations have been thought to be key factors in decreasing traffic injuries and deaths. the issue of human factors, such as drivers errors, seems to be a significant contributor to the rate of traffic injuries and deaths. the results of a study that was conducted in saudi arabia indicated that speed and non - compliance with rules were the main reasons for the high rate of injuries and deaths (6). a study of the knowledge, attitudes, and practices of 2200 drivers toward traffic regulations in tehran and zahedan indicated that the rate of road traffic crashes can be decreased by increasing the levels of knowledge of drivers and by changing their attitudes and practices. a significant association was found between safer attitudes and decreases in the rate of traffic crashes (or = 0.76, p = 0.007) (7). many observers have mentioned the importance of determining the relationships between personality and demographic variables (such as age, gender, and education levels) and drivers behaviors. in low - income countries, driving attitudes and behaviors intention to commit driving violations, high - speed driving, and poor decision making are important risk factors for traffic accidents (9). some previous studies have reported a weak association between knowledge, attitudes toward driving, and driving behavior (10). various surveys, such as the one conducted by yunesian and moradi, have shown that 67.7, 56.4, and 47.7% of drivers had adequate knowledge, positive attitudes, and safe practice towards traffic regulations, respectively. their findings suggested that the type of automobile, education levels, occupation, and marital status had significant effects on drivers practices. the researchers conclude that drivers in teheran city had risky practices towards traffic regulations (11). although, there has been considerable debate concerning whether attitudes predict behavior, several research studies have indicated that attitudes do, in fact, affect behavior. changing people s attitudes towards traffic regulations has been considered to be a key element in the prevention of traffic crashes (12). developing a culture of safety is the most effective strategy to prevent breaking the rules (13). some studies have shown that the use of seat belts by drivers reduces the risk of fatal injuries and reduces the severity of the effects of accidents on the occupants of vehicles. the most common causes of car accidents in malaysia were high - speed driving, dangerous driving, and the careless overtaking of other vehicles, i.e., with these causes contributing to 32.8, 28.2, and 15.1% of all accidents, respectively. the major cause of traffic accidents was drivers behaviors (76.1%) (14). traffic accidents are the main causes of disability - adjusted life years (dalys) in iran. more than 30,000 deaths due to traffic accidents are reported in iran each year, with an associated fatal incidence rate of 44 per 100,000) (15). the estimated cost of traffic injuries in iran in 2012 was 180,000 billion iranian rails (us$ 6,000,000,000), which amounted to 6.64% of iran s gross national income in 2013 (16). however, to date, there has been little discussion about the rate of traffic injuries and the related costs in bandar - abbas. the objectives of this research were to study the knowledge, attitudes, and practices of taxi drivers towards traffic regulations in bandar - abbas, iran, and to determine the relationships between demographic features and the knowledge, attitudes, and practices of drivers towards traffic regulations. this cross - sectional study was conducted in 2014 in bandar - abbas county, hormozgan province, iran. according to some cross - sectional studies performed in bandar - abbas on automobile drivers (17, 18), there is a need for more research on drivers behaviors in this city to reduce the severity of accidents and the fatality rates. we used the following formula to determine the sample size : n = z1-/2 p(1-p)/d, where n is the required sample size, z1-/2 is the value for a level of confidence (1.96), p is the expected proportion (0.5), and d is the precision (0.04). based on this formula, a sample size of 306 male taxi drivers was determined. another 43 drivers worked for exclusive taxi services and said they did not have time to participate in the research. thus, a total of 241 drivers (response rate 78.7%) who operated within the city of bandar - abbas participated in this study, and the data obtained from these drivers were used for the analyses. to study the knowledge, attitudes, and practices of intra - city drivers towards traffic regulations in bandar - abbas, the two authors designed questionnaires to assess their knowledge and attitudes and a checklist for assessing the drivers practices were used. the knowledge questionnaire included 15 questions extracted from parts of iran s driver s license test. the internal consistency of the knowledge questionnaire was assessed by cronbach s alpha (= 0.82). the attitude questionnaire included seven questions that were designed to assess drivers attitudes in relation to various traffic regulations. it was prepared under the supervision of experienced traffic police officers, and it included various items, such as using seat belts, exceeding the speed limit in low - volume traffic on intercity roads, driving in restricted areas in an emergency situation, keeping a safe distance behind the car in front, crossing the center line of a road in low - volume traffic, coming to a complete stop before entering a main street from a side street, and eating and drinking while driving. the internal consistency of the attitude questionnaire was assessed by cronbach s alpha (= 0.75). this checklist included 35 questions concerning drivers behaviors, such as risky overtaking maneuvers, exceeding the speed limit over speed bumps, turning in prohibited areas, sudden braking, and running a red light. chicago, il, usa) was used to analyze the data. the mean and standard deviation (sd) were used to report the numerical data, while frequency and percentage were used to report qualitative variables. the chi - squared test was performed to determine the relationships between the knowledge, attitudes, and practices of taxi drivers towards traffic regulations and demographic features. this cross - sectional study was conducted in 2014 in bandar - abbas county, hormozgan province, iran. according to some cross - sectional studies performed in bandar - abbas on automobile drivers (17, 18), there is a need for more research on drivers behaviors in this city to reduce the severity of accidents and the fatality rates. we used the following formula to determine the sample size : n = z1-/2 p(1-p)/d, where n is the required sample size, z1-/2 is the value for a level of confidence (1.96), p is the expected proportion (0.5), and d is the precision (0.04). based on this formula, a sample size of 306 male taxi drivers was determined. another 43 drivers worked for exclusive taxi services and said they did not have time to participate in the research. thus, a total of 241 drivers (response rate 78.7%) who operated within the city of bandar - abbas participated in this study, and the data obtained from these drivers were used for the analyses. to study the knowledge, attitudes, and practices of intra - city drivers towards traffic regulations in bandar - abbas, the two authors designed questionnaires to assess their knowledge and attitudes and a checklist for assessing the drivers practices were used. the knowledge questionnaire included 15 questions extracted from parts of iran s driver s license test. the internal consistency of the knowledge questionnaire was assessed by cronbach s alpha (= 0.82). the attitude questionnaire included seven questions that were designed to assess drivers attitudes in relation to various traffic regulations. it was prepared under the supervision of experienced traffic police officers, and it included various items, such as using seat belts, exceeding the speed limit in low - volume traffic on intercity roads, driving in restricted areas in an emergency situation, keeping a safe distance behind the car in front, crossing the center line of a road in low - volume traffic, coming to a complete stop before entering a main street from a side street, and eating and drinking while driving. the internal consistency of the attitude questionnaire was assessed by cronbach s alpha (= 0.75). this checklist included 35 questions concerning drivers behaviors, such as risky overtaking maneuvers, exceeding the speed limit over speed bumps, turning in prohibited areas, sudden braking, and running a red light. chicago, il, usa) was used to analyze the data. the mean and standard deviation (sd) were used to report the numerical data, while frequency and percentage were used to report qualitative variables. the chi - squared test was performed to determine the relationships between the knowledge, attitudes, and practices of taxi drivers towards traffic regulations and demographic features. tables 1 and 2 illustrate the questions about attitudes and practices and the percentage and frequency of drivers responses to attitude and practice items towards traffic regulations, respectively. the highest number of participants (55 drivers) was in the 3135 age group. two hundred thirty - eight of them (98.8%) were married. among all of the drivers, 113 obtained their driver s license between 1992 and 2002. among the 241 drivers, 97 (40.2%) had completed the middle school educational level, 32.4% of them had work experience of 610 years. as shown in tables 1 and 2, 120 drivers (49.8%) totally agreed with the statement, eating and drinking while driving is dangerous. and 124 of them (51.5%) believed that it is necessary to keep a safe distance from a car in front. one hundred and seven taxi drivers did not wear a seat belt while driving and 190 of them (78.8%) exchanged taxi fares while driving. there were no significant relationships between age, educational levels, and the year the drivers obtained their driver s licenses and drivers knowledge (p > 0.05). the chi - squared test showed a significant difference between knowledge and the work experience of drivers (p = 0.014). approximately 30.2% of the drivers (among 169 drivers) who had 610 years of work experience had adequate knowledge about traffic regulations. the results, as shown in table 5, indicated that there were no significant relationships between demographic features and the attitudes of drivers towards traffic regulations (p > 0.05). approximately 43.5% of the drivers in the 3140 year age group had positive attitudes towards traffic regulations (among 216 drivers expressed positive attitudes), while 46.8% of the drivers in this group who had diplomas and higher levels of education had positive attitudes. among the drivers who obtained their driver s licenses between 1992 and 2002, the data in table 6 indicate that there were no significant differences between the drivers practices and their demographic features (p > 0.05). ninety two drivers (44.4%) in the 3140 age group had safe practices towards traffic regulations (among 207 drivers with safe practice). in the group that had 6 to 10 years of work experience, 71 drivers (34.3%) had safe practices towards traffic regulations. the objectives of the current study were to study the knowledge, attitudes, and practices of taxi drivers towards traffic regulations in bandar - abbas, iran, and to determine the relationships between demographic features and knowledge, attitudes, and practices of drivers towards traffic regulations. ninety - seven drivers (40.6%) disagreed that the use of a seat belt caused discomfort. the importance of the wearing a seat belt has been mentioned in many previous studies (6, 7). the findings of many studies have shown that wearing a seat belt may decrease the risk and severity of injuries. although most of the drivers in their study (88%) were aware of the effectiveness and benefits of wearing a seat belt in decreasing the severity of injuries in traffic crashes, only 63% of them actually wore a seat belt while driving (19). approximately 15.8% of the drivers admitting to running red lights, and 44.4% of them performed risky overtaking maneuvers, both of which increase the risks of traffic accidents (20). most of the drivers believed that engaging in distracting activities, such as eating and drinking is dangerous while driving (49.8% totally agree and 34.9% agree). the results of other studies have indicated that distracting activities may decrease the driver s performance (21). among the 241 drivers in the current study, 103 of the drivers (42.7%) used a mobile phone while driving. among 287 victorian drivers (australian state of victoria), 60% of them used mobile phone while driving, and one - third of them used their phone in the hand - held mode (22). also, the results of some studies indicated that talking on a cell phone may increase the risks of traffic accidents (23). the results obtained from the assessment of relationships between demographic features and drivers knowledge indicated that drivers in the 3140 age group had adequate knowledge of traffic regulations. the chi - squared test showed a significant difference between drivers knowledge and the work experience of taxi drivers. a greater knowledge of traffic regulations was reported in drivers in the group that had 610 years of work experience. eighty - two drivers (48.5%) with the educational levels of diploma and higher had greater knowledge of traffic regulations than the other drivers. the study of drivers knowledge, attitudes and practices in tehran illustrated a significant relationship between education levels and drivers knowledge (p= 0.02). no significant differences were found between other demographic features and drivers knowledge. in other words, although no significant differences were found between drivers knowledge and their educational levels in the current study, a greater knowledge towards traffic regulations was reported in 48.5% of drivers with higher educational levels. the chi - squared test did not show any significant differences between the attitudes of drivers towards traffic regulations and demographic features. the findings of yunesian and moradi indicated that there were significant differences between the attitudes of drivers and two demographic variables of drivers, i.e., age (p= 0.02) and marital status (p= 0.002). the most positive attitudes towards traffic regulations in this study (43.5%) were reported in taxi drivers in the 3140 age group (among 216 drivers expressed positive attitudes). the safer practices (44.4%) were reported in drivers in the 3140 year age group (among 207 drivers with safe practice), drivers with the educational levels of diploma and higher (45.9%), and drivers in the group that had 610 years of work experience. these findings were consistent with other research in which it was found that drivers with higher educational levels may have safer practices than those with lower educational levels (11). many of the taxi drivers in bandar - abbas had inadequate knowledge, less positive attitudes, and risky practices towards traffic regulations. the increase in knowledge, attitudes, and practices of taxi drivers towards traffic regulations may decrease the rate of traffic injuries and deaths. implementation of effective intervention programs may increase the taxi drivers knowledge, attitudes, and practices towards traffic regulations. a limitation of this study was that the relationships between the knowledge, attitudes, and practices of taxi drivers towards traffic regulations and traffic crashes were not examined. many taxi drivers in bandar - abbas had inadequate knowledge, less positive attitudes, and risky practices towards traffic regulations. implementation of effective intervention programs may increase the taxi drivers knowledge, attitudes, and practices towards traffic regulations.	introductiontraffic injuries are among the leading causes of death and disability in many countries. the knowledge, attitudes, and practice of drivers towards traffic regulations are key factors in decreasing traffic injuries and deaths. the objectives of this research were to study the knowledge, attitudes, and practice of taxi drivers towards traffic regulations in bandar - abbas, iran, and to determine the relationships between demographic features and knowledge, attitudes, and practice of taxi drivers towards traffic regulations.methodsthis cross - sectional study was done in 2014 in bandar - abbas, iran (hormozgan province). to study the knowledge, attitudes, and practice of 241 intra - city taxi drivers towards traffic regulations, researchers developed questionnaires and a checklist. the chi - squared test was performed to determine the relationships between knowledge, attitude, and practice of drivers towards traffic regulations and demographic features.resultsamong the 241 drivers, 50 of them (20.7%) thought that the seat belt could cause discomfort while driving, and 107 (44.4%) did not wear a seat belt while driving. the study determined that there was a significant difference between the knowledge and work experience of the drivers (p = 0.014). the 94 drivers (43.5%) in the 3140 year age group had positive attitudes towards traffic regulations (among 216 drivers expressed positive attitudes) and 92 (44.4%) of the drivers in this age group had safe practices towards traffic regulations (among 207 drivers with safe practice).conclusionmany of the taxi drivers in bandar - abbas had inadequate knowledge, less positive attitudes, and risky practices towards traffic regulations. implementation of effective intervention programs may increase the taxi drivers knowledge, attitudes, and practices towards traffic regulations.
human metcam (humetcam), a cam in the immunoglobulin - like gene superfamily, is an integral membrane glycoprotein. alternative names for metcam are muc18, cd146, mcam, melcam, a32, and s - endo 1. to avoid confusion with mucins and to reflect its biological functions, we have renamed muc18 as metcam (metastasis cam), which means an immunoglobulin - like cam that affects or regulates metastasis,. the humetcam has 646 aminoacids that include a n - terminal extracellular domain of 558 aminoacids, which has 28 aminoacids characteristics of a signal peptide sequence at its n - terminus, a transmembrane domain of 24 aminoacids (amino acid number 559583), and a cytoplasmic domain of 64 aminoacids at the c - terminus. humetcam has eight putative n - glycosylation sites (asn - x - ser / thr), of which six are conserved, and are heavily glycosylated and sialylated resulting in an apparent molecular weight of 113,000150,000. the extracellular domain of the protein comprises five immunoglobulin - like domains (v - v - c2-c2-c2) [1, 7 ] and an x domain. the cytoplasmic tail contains peptide sequences that will potentially be phosphorylated by protein kinase a (pka), protein kinase c (pkc), and casein kinase 2 (ck 2) [1, 7, 8 ]. my lab has also cloned and sequenced the mouse metcam (mometcam) cdna, which contains 648 aminoacids with a 76.2% identity with humetcam, suggesting that mometcam is likely to have biochemical properties and biological functions similar to the human counterpart. the structure of the humetcam protein is depicted in figure 1, suggesting that metcam, similar to most cams, plays an active role in mediating cell - cell and cell - extracellular interactions, crosstalk with many intracellular signaling pathways, and modulating the social behaviors of cells. humetcam is expressed in a limited number of normal tissues, such as hair follicular cells, smooth muscle cells, endothelial cells, cerebellum, normal mammary epithelial cells, basal cells of the lung, activated t cells, intermediate trophoblast, and normal nasopharyngeal epithelial cells. the protein is overly expressed in most (67%) malignant melanoma cells, and in most (more than 80%) premalignant prostate epithelial cells (pin), high - grade prostatic carcinoma cells, and metastatic lesions [12, 13 ]. humetcam is also expressed in other cancers, such as gestational trophoblastic tumors, leiomyosarcoma, angiosarcoma, haemangioma, kaposi 's sarcoma, schwannoma, some lung squamous and small cell carcinomas, some breast cancer, some neuroblastoma, and also nasopharyngeal carcinoma and ovarian cancer. it is now well documented that in addition to tissue - specific signatures in different cancer types, cancers from different tissues also express some common genes [1517 ]. cams do not merely act as a molecular glue to hold together homotypic cells in a specific tissue or to facilitate interactions of heterotypic cells ; cams also actively govern the social behaviors of cells by affecting the adhesion status of cells and modulating cell signaling. they also actively mediate the cell - to - cell and cell - to - extracellular matrix interactions to allow cells to constantly respond to physiological fluctuations and to alter / remodel the surrounding microenvironment for survival. they do so by crosstalk with cellular surface growth factor receptors, which interact with growth factors that may be secreted from stromal cells or released from circulation and embedded in the extracellular matrix [18, 19 ]. thus, an altered expression of cams affects the motility and invasiveness of many tumor cells in vitro and metastasis in vivo [18, 19 ]. cams also play an important role in the favorable soil that provides a proper microenvironment at a suitable period to awaken the dormant metastatic tumor cells to enter into an aggressive growth phase. actually, the metastatic potential of a tumor cell, as documented in many carcinomas, is the consequence of a complex participation of many over- and under - expressed cams [18, 19 ]. based on the above information, aberrant expression of humetcam may also affect the motility and invasiveness of many tumor cells in vitro and metastasis in vivo. it is logical to hypothesize that humetcam / muc18 should play an important role in promoting the malignant progression of many cancer types [7, 18 ]. however, recently we observed an unexpected opposite function of metcam / muc18 in the malignant progression of a mouse melanoma subline and ovarian cancer cells, in which it functioned as a tumor and metastasis suppressor (wu, unpublished results). in this paper, we will review its dual roles in the tumorigenesis and metastasis in different cancer types. metcam - induced tumorigenesis has been studied in melanoma, prostate cancer, breast cancer, and ovarian cancer. overexpression of metcam may have no effect, a negative effect, or a positive effect on tumorigenesis, dependent upon the cell lines used, as shown in the following. overexpression of metcam had a slight tumor suppression effect on tumorigenesis of human melanoma cells in xenograft mice, as shown in figure 2, but it had no effect on tumorigenesis of two sublines, number 3 and number 10, of the mouse melanoma cell line k1735 in syngeneic mice. figure 3 shows only the effect of mometcam on the tumorigenesis of k1735 - 3. only one group showed that overexpression of metcam increased tumorigenesis of a human melanoma cell line in xenograft mice ; however, the results were questionable because only the tumorigenicity of one mouse injected with metcam - expressing clone and one mouse with control cells was determined, and thus no standard deviations were indicated and no statistical analysis was done, as shown in figure 4. the most compelling evidence for its tumor suppressor effect is in the subline number 9 of the mouse melanoma cell line k1735 (k9) in syngeneic c3h mice. overexpression of mometcam in the k9 cells decreased subcutaneous tumorigenesis in immunocompetent syngeneic c3h mice [22, 23 ], as shown in figure 5. shih. showed that metcam was not expressed in mcf-7 cell line, and they showed that the overexpression of humetcam in mcf-7 cells suppressed tumor formation of the cells in scid mice, as shown in figure 6, suggesting that metcam is a possible tumor suppressor in breast cancer. we have confirmed from their western blot and immunohistochemistry results that metcam is not expressed in mcf-7 cells (0%), very weakly expressed in sk - br-3 cells (5%), and weakly expressed, though slightly higher levels than the above two cells lines, in the human mammary cancer cell lines, mda - mb-231 (a low metastatic cell line) (16%), and mda - mb-468 (a high metastatic cell line) (22%), as shown in figure 7. recently gene expression profiles of breast cancer cell lines have indicated that the gene expression profiles of mcf-7 and sk - br3 are more closely related to the luminal subtype of the breast cancers, whereas those of mda - mb-231 and mda - mb-468 are more closely related to the basal - like subtype [25, 26 ]. it appeared that metcam is not or weakly expressed in cell lines established from luminal subtypes, but it is moderately expressed in cell lines established from basal - like subtypes, mda - mb-231 and mda - mb-468.. found that overexpression of metcam inhibited the in vitro invasiveness of mda - mb-231 cells, supporting the notion of shih. on the contrary, garcia. and zabouo. supported the opposite role of metcam in the progression of human breast cancer cells in that it plays a role of tumor promoter. however, all three groups did not substantiate their claim with studies in animal models. to resolve this controversy, we recently reinvestigated the role of metcam in the tumorigenesis of human breast cancer cells in animal models and found that overexpression of metcam promoted the tumorigenesis of four human breast cancer cell lines, mcf7, sk - br-3, mda - mb-231, and mda - mb-468 [30, 31 ]. tumorigenesis of mcf-7 in female scid mice is shown in figure 8, and that of sk - br-3 in female nude mice in figure 9. thus, the tumor suppression role of metcam in tumorigenesis of human breast cancer cells is not supported by the above evidence. on the contrary, it suggests the alternative notion that metcam increased tumorigenesis and perhaps also the metastasis of human breast cancer cells. recently, both our group and another group found that metcam was upregulated in human ovarian cancer specimens, suggesting that metcam may be a marker for the poor prognosis of ovarian cancer patients [14, 32 ], and that metcam may play a positive role in the development of ovarian cancer [14, 32 ]. however, preliminary animal tests (injection of bg-1 cells in nonorthotopic, subcutaneous sites of female nude mice) show that overexpression of metcam did not have any significant effect on the tumor formation of a human ovarian cancer cell line, bg-1 (data not shown). to rule out the possibility that this effect might be an artifact because the tests were carried out in the nonorthotopic, subcutaneous sites, which did not provide a proper microenvironment for tumorigenesis, we carried out further tests of the effect of overexpression of metcam on tumorigenesis of bg-1 cells by injecting the clones in an orthotopic site, the intraperitoneal cavity of female scid mice. we found that tumorigenesis of bg-1 clones was also very poor, suggesting that estrogen supplement by subcutaneous implantation may enhance the tumorigenesis of bg-1 cells in both immunodeficient mice. nevertheless, the test of the effect of overexpression of metcam on tumorigenesis of bg-1 cells in orthotopic sites had a somewhat suppressive effect, as shown in figure 10. we also carried out animal studies by using another human ovarian cancer cell line, sk - ov-3 and found that overexpression of metcam suppressed tumorigenesis of sk - ov-3 cells at both nonorthotopic, subcutaneous sites, as well as an orthotopic site (the intraperitoneal cavity), as shown in figure 11. overexpression of metcam significantly increases the tumor - take and promote tumorigenicity and tumorigenesis of a human prostate cancer cell line, lncap, as shown in figure 12 [35, 36 ]. we found that mometcam was expressed at a higher level in a mouse angiosarcoma clone, svr, which was transfected with h - ras, than in the immortalized normal endothelial cell line control, ms-1 (figure 13). the higher level of mometcam expression appeared to correlate with the higher tumorigenicity of the svr cell line [7, 37 ], suggesting a positive role for metcam in promoting angiosarcoma. there is a negative correlation of metcam expression with the human nasopharyngeal carcinoma specimens, suggesting that metcam may also play a tumor suppressor role in the tumorigenesis of nasopharyngeal carcinoma. a tumor suppressor role of metcam may also be implicated in haemangiomas, since metcam expression was decreased during the progression of haemangiomas. metcam - induced metastasis has been studied in melanoma, prostate cancer, osteosarcoma, and ovarian cancer lines. overexpression of metcam in melanoma cells mostly have a positive effect on the metastasis of human melanoma cell lines in immunodeficient mice (both athymic nude and scid mice) [3, 20 ], mouse melanoma cell lines in syngeneic mice [7, 21 ], and a human prostate cancer cell line, lncap, in nude mice [7, 13, 36 ]. overexpression of metcam also has a positive effect on the metastasis of osteosarcoma cell lines. surprisingly, we have recently found that overexpression of metcam has a negative effect on the metastasis of one subline, number 9, of mouse melanoma cell k1735 [22, 23 ] and ovarian cancer cell lines [3032 ]. humetcam was originally found to be abundantly expressed on the cellular surface of most malignant human melanomas ; since then, it has been postulated to play a role in the progression of human melanoma. this notion is also supported by the positive correlation of mometcam expression with the metastatic ability of several mouse melanoma cells lines. definitive proof comes from the results that the stably ectopic expression of the humetcam cdna gene in three nonmetastatic human cutaneous melanoma cell lines increases the metastatic abilities of these cell lines in immune - deficient mouse models [3, 20 ]. furthermore, the stable, ectopic expression of mometcam cdna in two low - metastatic mouse melanoma cell lines increases the metastatic abilities of these cell lines in immune - competent syngeneic mice. however, metcam enables melanoma cells to establish pulmonary metastasis only when the cells are injected into the tail vein (experimental metastasis assay) [3, 20, 21 ], thus bypassing the initial stages of metastasis. no metastasis was found when metcam - expressing melanoma cells were injected subcutaneously (spontaneous metastasis assay) either in immune - deficient mouse models [3, 20 ] or in immune - competent syngeneic mouse models. taken together, metcam definitely promotes the metastasis of melanoma cells, but at later stages ; thus overexpression of metcam did not initiate the metastasis of melanoma cells. this result is consistent with the recent observation that fibroblast growth factor 2, but neither humetcam nor integrin actually initiates the malignant progression of subcutaneous melanocyte into melanoma. in contrast to these results, overexpression of mometcam in one mouse melanoma cell line k1735 subline number 9 (k9) decreased pulmonary lung nodule formation when cells were injected into tail veins (experimental metastasis test) [22, 23 ], as shown in figure 14. overexpression of metcam is not limited to melanoma as previously thought [7, 10 ]. our group has pioneered the successful determination of humetcam expression in prostate cancer cells and tissues using our chicken polyclonal anti - humetcam and carried out extensive studies of humetcam - mediated prostate cancer metastasis. we have used molecular biological and immunological methods to study the expression of humetcam in three established prostate cancer cell lines and human prostate cancer tissues, and in immunohistochemical studies of paraffin - embedded human prostate cancer tissue sections [7, 8, 12, 13 ]. from the results, we have suggested that humetcam may be a new diagnostic marker for the metastatic potential of human prostate cancer. this is further corroborated by results of a positive correlation of mometcam expression with the progression of mouse prostate adenocarcinoma in a transgenic mouse model, tramp. from these results, we have also suggested that humetcam may be a key determinant in promoting tumorigenesis and metastasis of human prostate cancer cells. to test this hypothesis, we determined the effect of ectopic expression of humetcam on the ability of human prostate lncap cells to form tumor in the prostate gland and to initiate metastasis in nude mice. we found that overexpression of metcam had a positive effect on the metastasis of the human prostate cancer cell, lncap, when the cells were injected at the orthotopic site (the dorsolateral lobes of the nude mice). the metastatic lesions were found in multiple organs, such as seminal vesicles, ureter, kidney, and periaortic lymph nodes. different mice had metastatic lesions in one or two organs, but all of them had metastatic lesions in the lymph nodes. the parental lncap cells, which do not express any metcam, can form tumors in the prostate, but these tumors did not manifest any metastasis. but our recent preliminary results appear to show that overexpression of metcam may be able to enhance establishment of the growth of a bone - homing c42b clone of lncap cells in nude mice., metcam can actually initiate the metastasis of lncap cells, thus affecting the progression of prostate cancer cells at the early stage of metastasis [7, 36 ]. recently, one group has shown that metcam is overly expressed in two of the six human osteosarcoma cell lines. the metastasis can be blocked by a humanized antibody against metcam, suggesting metcam plays a positive role in the progression of osteosarcomas. recently we found that overexpression of metcam / muc18 suppressed metastasis and ascites formation of sk - ov-3 cells in the intraperitoneal cavity, as shown in figure 15. decreased expression of metcam has been correlated with the progression of haemangioma, suggesting the possible negative effect of metcam on progression of haemangioma. though metcam was downregulated in nasopharyngeal carcinoma, interestingly it was upregulated again in metastatic lesions in nasopharyngeal patients, suggesting that metcam may play a positive role in the malignant progression of nasopharyngeal carcinoma after a transient suppression of tumorigenesis. taken together, we suggest that the possible tumor and metastasis suppressor role of metcam may not be limited to melanoma and ovarian cancers, and that this may be a new function of metcam yet to be explored. summary 3.5 table 1 summarizes the possible role of metcam in the tumorigenesis and metastasis of various tumors / cancers.taken together, humetcam is a tumor promoter for prostate and breast cancers, and a metastatic gene for most melanoma cell lines, prostate cancer, osteosarcoma, and perhaps, breast cancer and nasopharyngeal carcinoma. it is a tumor suppressor for a mouse melanoma subline and ovarian cancers, and perhaps, haemangioma and nasopharyngeal carcinoma ; it is a metastasis suppressor for a mouse melanoma subline, ovarian cancer, and perhaps, haemangioma. table 1 summarizes the possible role of metcam in the tumorigenesis and metastasis of various tumors / cancers.taken together, humetcam is a tumor promoter for prostate and breast cancers, and a metastatic gene for most melanoma cell lines, prostate cancer, osteosarcoma, and perhaps, breast cancer and nasopharyngeal carcinoma. it is a tumor suppressor for a mouse melanoma subline and ovarian cancers, and perhaps, haemangioma and nasopharyngeal carcinoma ; it is a metastasis suppressor for a mouse melanoma subline, ovarian cancer, and perhaps, haemangioma. how does metcam mediate or regulate tumorigenesis and metastasis of cancer cells ? by deducing knowledge learned from the tumorigenesis of other tumors [1519, 42 ] and the humetcam - mediated progression of melanoma [4345 ] and angiogenesis [2, 4651 ], we may be able to find some common clues to begin understanding its mechanisms. first, the transcriptional expression of metcam gene may be regulated by pka / creb (camp - responsive element binding protein), ap-2 [44, 45 ], and other transcription factors, such as sp-1, c - myb, n - oct2, ets, carg, egr-1, and transcription factors binding to insulin - response elements, as shown in figure 16. among these potential regulators, it is well documented that the ap-2 transcription factor plays a crucial tumor suppressor role in the progression of melanoma, prostate, and breast cancer. it has been shown that pka / creb plays a positive role in the progression of melanoma, and perhaps also applicable to breast cancer and prostate cancer, by inhibiting the expression of ap-2and increasing the expression of metcam. the roles of other transcription regulators, tissue - specific enhancers and repressors, epigenetic control, and control at the level of chromatin remodeling of the gene have still yet to be investigated. second, since the cytoplasmic tail of metcam contains consensus sequences potentially to be phosphorylated by pka, pkc, and ck2, it may manifest its functions by crosstalk with various signaling pathways mediated by these protein kinases. for example, metcam expression in melanoma cells is reciprocally regulated by akt, in which akt up - regulates the level metcam and overexpression of metcam activates endogenous akt, which in turn inhibits apoptosis and increases survival ability. however, it is not clear if a similar mechanism is also used in breast, prostate, and other cancers. also, the detailed mechanism of how akt up - regulates the expression of metcam has not been worked out. pka, pkc, and ck2 may phosphorylate the cytoplasmic tail of metcam, which then facilitates its interaction with fak, thus promoting cytoskeleton remodeling. alternatively, after phosphorylation of its cytoplasmic tail by these protein kinases, metcam may interact with the downstream effectors of ras, activating erk and jnk, which in turn may transcriptionally activate the expression of akt or other genes that promote the proliferation and angiogenesis of tumor cells. though metcam has not been shown to be a substrate of ck2, which has been shown to phosphorylate other cams, such as cd44, e - cadherin, l1-cam, and vitronectin, it is also likely that ck2 may be able to phosphorylate metcam and link it to akt to affect the proliferation, survival, and other tumorigenesis - related functions of tumor cells. third, after the engagement of metcam with the ligand(s) or extracellular matrix, it may transmit the outside - in signals into tumor cells by activating fak and the downstream - signaling components, promoting cytoskeleton remodeling and increasing tumor cell motility and invasiveness [2, 7 ]. fourth, from what we know about the roles of other cams in the progression of other tumors [1519, 42 ], it is logical to postulate that metcam may affect cancer cell progression by crosstalk with signaling pathways that affect apoptosis, survival and proliferation, and angiogenesis of tumor cells [7, 18, 42 ]. thus, metcam may affect tumorigenesis and metastasis by altering the expression of various indexes in apoptosis, survival signaling, proliferation signaling, and angiogenesis. to support this notion, we have found that metcam promotes the progression of prostate cancer cells by rendering the cells with increased proliferative ability by elevating levels of ki67 and pcna, with increased survival ability by elevating the level of phosphorylated akt, and with increased angiogenic ability by elevating levels of vegf, vegfr2, and cd31 ; but it has no effect on the process of apoptosis. in contrast to this, metcam promotes the progression of melanoma cells differently by preventing the apoptosis of melanoma cells and reciprocally affecting the expression of a survival index, phospho - akt. further systematic studies by using specific rnais to knockdown the downstream effectors one - by - one in the metcam - expressing clones may be necessary to further understand this aspect of mechanism. fifth, metcam may mediate hematogenous spreading of melanoma cells, which had been implicated by its expression in endothelial cells, as well as in malignant melanoma cells, further shown to be present in the junctions of endothelial cells [49, 50 ] and essential for tumor angiogenesis in at least three tumor cell lines and human prostate cancer lncap cells. it is highly likely that metcam expression may promote hematogenous spreading of prostate cancer cells, similar to melanoma cells. similar mechanisms may also be used for the metcam - mediated hematogenous spreading of breast cancer and osteosarcoma cells. however, it is not known if metcam also plays a role in the lymphatic spread of cancer cells. recent results from one group showed that metcam is one of the lymphatic metastasis - associated genes, which is upregulated in malignant mouse hepatocarcinoma ; suggesting that metcam may also play a role in promoting lymphatic metastasis of cancer cells. labeling the cells with viable dyes and following the process in real time by using a newly developed nonintruding, but highly photo - penetrating imaging method of photoacoustic tomography (pat) [54, 55 ] may be useful for monitoring each step in the metcam - mediated progression. for the metcam - mediated dynamic spreading of melanoma cells in vivo, the pat imaging method coupled with using hairless syngeneic mouse animal models should reveal more clearly the process in real time. sixth, metcam has been shown to express in normal mesenchymal cells (smooth muscle, endothelium, and schwann cells) in the tissue stroma and be a marker for the mesenchymal stem cells, metcam may play an important role in regulating tumor dormancy or awakening, driving or preventing cancer cells to premetastatic niche, and formatting a microenvironment for favorable or unfavorable tumor growth in secondary sites. seventh, metcam may affect the progression of cancer cells by interactions with the host immune system, which, however, has been shown to have a paradoxical role in tumor progression. recently, one group has shown that a subset of host b lymphocytes may control melanoma metastasis through metcam - dependent interaction. on the other hand, it is highly likely that the tumor suppression effect of metcam expression in melanoma k1735 - 9 subline may be due to the interaction of metcam - expressing cells with the host immune defense system in the immunocompetent syngeneic c3h brown mouse, since the intrinsic motility and invasiveness of mouse melanoma k1735 - 9 was increased by the metcam expression [22, 23 ]. for example, the surface metcam expressed in this particular melanoma cell line may have a homophilic interaction with the nk cells, which also express metcam and enhance cytotoxic functions of nk cells. this hypothesis should be testable by studying the metcam - mediated metastasis of metcam - expressing k1735 - 9 cells in various genetically altered mice with a knockout of cd4 + t cells, cd8 + t cells, or nk cells, or mice with a combined knockout of these immune cells. eighth, malignant progression of cancer cells has been shown to associate with an abnormal glycosylation, resulting in expression of altered carbohydrate determinants. thus, the glycosylated status of metcam in different cancer types may be different from normal cells, thus manifesting positive or negative effect on the progression of different cancer types. this aspect of the metcam - mediated cancer progression has not been well studied, but is especially intriguing since metcam possesses six conserved n - glycosylation sites in the extracellular domain [7, 8 ]. we should always keep in mind that mechanisms of metcam - mediated cancer progression may be slightly different in different cancer cells due to their different intrinsic properties, which provides different cofactors and/or different ligand(s) that either positively or negatively regulate the metcam - mediated tumorigenesis and metastasis. to further understand the role of metcam in these processes, it is essential to diligently identify the cofactors and the metcam - cognate heterophilic ligand(s), which modulate the biological functions of metcam. the endeavor in this direction appears to be promising from our preliminary attempts that we may have successfully found a possible candidate of metcam 's heterophilic ligand in metcam - expressing human prostate cancer cells. mechanisms of metcam - mediated negative role in the progression of some cancer cells have not been studied at all. does metcam in some cancers behave like e - cadherin, which always plays a negative role in the tumorigenesis and metastasis of most epithelial cancer cells ? but even e - cadherin may function differently in different cancer cells. for example, its expression is temporally different and correlates with different stages during the progression of ovarian cancer : e - cadherin is not expressed in the ovarian surface epithelial cells, expressed in premalignant lesions and in well - differentiated tumors, and finally not expressed in late - stage invasive tumors. likewise, metcam may express and function normally in the normal nasopharyngeal epithelium, transiently reduce its expression and lose its function during the development of nasopharyngeal carcinoma, resume its expression, and function in the invasion stage of the cancer. alternatively, metcam may behave differently from e - cadherin by being modulated by different cofactors or ligands, which are expressed at different stages of the cancer. the tumor suppressor role of metcam in ovarian cancer cells may not be due to the altered intrinsic properties of the cancer cells, since the intrinsic motility and invasiveness of human ovarian cancer bg-1 and sk - ov-3 cells was not affected by the metcam expression [34, 35 ]. our preliminary results appear to suggest a special mechanism that a soluble form of metcam, which is produced by mmps in the metcam - expressing cells, may mediate the suppressive effect in ovarian cancer cells, similar to the production of a soluble form of p - cadherin by the induced mmps in breast cancer cells, which then dictates, instead of suppresses, the aggressive behavior of the breast cancer cells. metcam may have a key positive function in the progression of angiosarcoma, breast cancer, osteosarcoma, prostate cancer, and most melanoma cell lines. on the other hand, it may also have a key function in suppressing the progression of a few melanoma cell lines, ovarian cancer, haemangioma, and other cancers. to further understand its mechanisms in these processes, it is crucial to define its functional domains, identify its cognate ligand(s) and cofactor regulators, and study its crosstalk with members of various signaling pathways. these model systems may be useful for real - time observation of the dynamic process of cancer progression by using a nonintrusive and high photo - penetrating imaging system, such as the newly developed photoacoustic tomography (pat), to further understanding the process in mouse models [54, 55 ]. the knowledge gained would also be useful for designing effective means to decrease, or even to block the metastatic potential of these cancers. along these lines, a preclinical trial of using doxazosin, an 1-adrenergic antagonist that has been used to treat the bph patients, has been shown to be able to suppress prostate cancer metastasis in the tramp mouse model. furthermore, preclinical trials using a fully humanized anti - metcam antibody against melanoma growth and metastasis [65, 66 ] and using a mouse anti - metcam monoclonal antibody against angiogenesis and tumor growth of hepatocarcinoma, leiomyosarcoma, and pancreatic cancer have been successfully demonstrated. alternatively, small soluble peptides derived from metcam may also be useful for blocking the tumor formation and tumor angiogenesis [52, 67, 68 ].	metcam, an integral membrane cell adhesion molecule (cam) in the ig - like gene superfamily, is capable of performing typical functions of cams, such as mediating cell - cell and cell - extracellular interactions, crosstalk with intracellular signaling pathways, and modulating social behaviors of cells. metcam is expressed in about nine normal cells / tissues. aberrant expression of metcam has been associated with the progression of several epithelial tumors. further in vitro and in vivo studies show that metcam plays a dual role in the progression of different tumors. it can promote the malignant progression of several tumors. on the other hand, it can suppress the malignant progression of other tumors. we suggest that the role of metcam in the progression of different cancer types may be modulated by different intrinsic factors present in different cancer cells and also in different stromal microenvironment. many possible mechanisms mediated by this cam during early tumor development and metastasis are suggested.
cutaneous leishmaniasis (cl) which is associated with considerable morbidity is still a major health problem in the world especially in developing countries. over the years evidence - based studies are few and it is difficult to determine what the best treatments are.1 for many decades, pentavalent antimonials have remained the gold standard of treatment. in a recent study, a meta - analysis of treatment of new world cutaneous leishmaniasis showed a 76.5% cure rate in 1150 patients treated with pentavalent antimonials and concluded that the pentavalent antimonials were as effective as pentamidine.2 in the most recent studies, cure rates have not gone beyond 80%.3 several factors, most important of which are the strain of leishmania and host 's immune system, determine the biologic behaviour of the disease. in assessing the efficacy of treatment, the natural course of the disease has to be taken into consideration as well as the fact that cl often heals spontaneously within one week to three years, usually within one year. to be considered also when the best mode of treatment is being determined are the cost, availability of drugs, adverse effects and the local experience. over the last 50 years, though only a few drugs for cutaneous leishmaniasis have emerged, drug resistance has increased. in this review, the newly introduced medications and those being developed will be discussed. newly introduced medications / procedures for treatment of cl amphotericin b is the standard drug for the treatment of systemic fungal infections. it has also been used as the second line of treatment for visceral leishmaniasis (vl) resistant to pentavalent antimonials. amphotericin b, however, has a low therapeutic index, high acute toxicity and necessitates parenteral administration. to avoid these drawbacks, a new lipid - associated formulation of the original polyene amphotericin b, liposomal amphotericin b has been introduced. this formulation is better tolerated and has less infusion - related toxicity and nephrotoxicity than amb, but has the same efficacy as the original formulation. it is administered intravenously in a dose 3 10 mg / kg / day for 5 7 days. the lower dosage and shorter duration are as effective as the higher dosages and longer duration of treatment. the recently introduced l- amb formulation has shown proven efficacy in old world and new world vl (owvl and nwvl), 45 however, its use in old and new world cl has been limited. other formulations such as amph b colloidal dispersion (abcd) and amph b lipid complex (ablc) have been developed. these have been investigated primarily for the treatment of invasive fungal infections and are effective and less toxic than amph b. investigations of the use of these formulations for the treatment of leishmaniasis are limited, so more trials are needed. it was registered in 2002 in india for the treatment of visceral leishmaniasis and currently in use in the full range of clinical leishmaniasis.711 a review of pubmed articles from 2005 2008 dealing with the use of miltefosine in leishmaniasis shows that it is an effective recommended treatment for indian, ethiopian vl, and columbian and bolivian cl. it is also the drug of choice for unusual forms of leishmaniasis that require long periods of treatment as in diffuse cutaneous leishmaniasis (dcl) and post - kala - azar dermal leishmaniasis (pkadl).7 of special interest, is the study of the pharmacokinetics of miltefosine in the treatment of old world cl (owcl). in this recent study, miltefosine was given to 31 dutch military personnel who contracted leishmaniasis while serving in afghanistan, at a dose of 150 mg / day for 28 days with a maximum follow up duration of 202 days after initiation of treatment. the concentration of miltefosine was determined in the blood samples during the period of treatment and up to 5 months post treatment. results showed the first elimination half - life to be 7.05 days and the terminal elimination half - life of 30 days with a mean concentration of 30,800ng / ml in the last week of treatment (22 - 28 days). all analyzed samples contained concentrates of miltefosine above the lower limit of qualification (lloq) of 4ng / ml. miltefosine was detected in human plasma samples for up to 5 - 6 months after cessation of treatment. the implication of the detection of subtherapeutic concentrations of miltefosine in the blood beyond five months post treatment may indicate emerging resistant parasites as well as risks of teratogenicity.12 the efficacy and safety profile of miltefosine in the treatment of zoonotic cutaneous leishmaniasis (zcl) caused by l. major in iran has been studied and compared with meglumine antimoniate in a randomized open - label study. definitions of lesion cure and failure were based on both clinical and parasitological criteria two weeks after the end of treatment, and clinical recovery three months after the parasitological cure period. miltefosine was given to 32 subjects with zcl, while 31 received i m meglumine antimoniate in a dose of 20 mg sb (5) kg / daily for 14 days. of the 28 subjects who completed treatment with miltefosine, 26 were cured at 3 months corresponding to a 92.9% cure rate. there was one treatment failure (3.1%), one relapse (3.1%) and four drop - outs as a result of lack of tolerance of the drug. in the antimonial group, 25 of the 31 subjects were cured (83.3%) ; five failed (16.1%) and one (3.2%) was lost at three months of follow up. no relapses were observed in the group who received the i m meglumine antimoniate at six months follow - up after the cessation of treatment. both treatment protocols were tolerated but nausea (32.2%) and vomiting (21.5 %) were observed within the first two weeks of initiation of the oral miltefosine regimen. other gastrointestinal and musculoskeletal and total adverse reactions were not significant in the two groups. the study concluded that miltefosine was as effective as meglumine antimoniate in the treatment of zcl.13 this is a recently introduced topical immunomodulator initially approved for the treatment of genital warts. subsequently, it has been used in a wide spectrum of diseases, both inflammatory and neoplastic. in a study in peru, imiquimod cream 7.5% was used on alternate days for 20 days in a group with newly diagnosed cl. even though, there was an initial response, there was a relapse on discontinuation of treatment.14 photodynamic therapy is a recently introduced modality approved for the treatment of actinic keratoses. it involves the topical application of a porphyrin precursor, aminolevulinic acid (ala) or methyl - aminolevulinate (mal), followed by irradiation with laser or intense pulsed light (ipl). since its first introduction more recently, this therapeutic modality has been utilized in non - neoplastic conditions including cutaneous leishmaniasis.15 few mechanistic studies have addressed the principles underlying the use of pdt for the treatment of cl.1618 in vitro and mechanistic studies of ala - pdt against cl did not demonstrate any parasiticidal effects on amastigotes and no differences in ala - derived ppix levels were detected between leishmania - infected and non - infected j774.2 cells.16 in contrast, in vivo topical ala - pdt performed on a murine cl model resulted in extensive tissue destruction and significant reduction of parasite load. a dramatic decrease of macrophages and increased levels of interleukin six was observed in infected skin. these findings suggest that the parasiticidal effect of ala - pdt for cutaneous leishmaniasis is indirect and mediated through the killing of host cells rather than the direct destruction of parasites.1617 a review of six clinical studies investigating the use of pdt in 39 patients with a total of 77 lesions for the treatment of old world cl showed that pdt with the porphyrin precursors is relatively effective. however, the data is still limited and pdt can not be recommended in routine clinical practice at this point. even though it is considered a promising therapeutic modality, additional controlled trials and further investigations it has also been used as the second line of treatment for visceral leishmaniasis (vl) resistant to pentavalent antimonials. amphotericin b, however, has a low therapeutic index, high acute toxicity and necessitates parenteral administration. to avoid these drawbacks, a new lipid - associated formulation of the original polyene amphotericin b, liposomal amphotericin b has been introduced. this formulation is better tolerated and has less infusion - related toxicity and nephrotoxicity than amb, but has the same efficacy as the original formulation. it is administered intravenously in a dose 3 10 mg / kg / day for 5 7 days. the lower dosage and shorter duration are as effective as the higher dosages and longer duration of treatment. the recently introduced l- amb formulation has shown proven efficacy in old world and new world vl (owvl and nwvl), 45 however, its use in old and new world cl has been limited. other formulations such as amph b colloidal dispersion (abcd) and amph b lipid complex (ablc) have been developed. these have been investigated primarily for the treatment of invasive fungal infections and are effective and less toxic than amph b. investigations of the use of these formulations for the treatment of leishmaniasis are limited, so more trials are needed. it was registered in 2002 in india for the treatment of visceral leishmaniasis and currently in use in the full range of clinical leishmaniasis.711 a review of pubmed articles from 2005 2008 dealing with the use of miltefosine in leishmaniasis shows that it is an effective recommended treatment for indian, ethiopian vl, and columbian and bolivian cl. it is also the drug of choice for unusual forms of leishmaniasis that require long periods of treatment as in diffuse cutaneous leishmaniasis (dcl) and post - kala - azar dermal leishmaniasis (pkadl).7 of special interest, is the study of the pharmacokinetics of miltefosine in the treatment of old world cl (owcl). in this recent study, miltefosine was given to 31 dutch military personnel who contracted leishmaniasis while serving in afghanistan, at a dose of 150 mg / day for 28 days with a maximum follow up duration of 202 days after initiation of treatment. the concentration of miltefosine was determined in the blood samples during the period of treatment and up to 5 months post treatment. results showed the first elimination half - life to be 7.05 days and the terminal elimination half - life of 30 days with a mean concentration of 30,800ng / ml in the last week of treatment (22 - 28 days). all analyzed samples contained concentrates of miltefosine above the lower limit of qualification (lloq) of 4ng / ml. miltefosine was detected in human plasma samples for up to 5 - 6 months after cessation of treatment. the implication of the detection of subtherapeutic concentrations of miltefosine in the blood beyond five months post treatment may indicate emerging resistant parasites as well as risks of teratogenicity.12 the efficacy and safety profile of miltefosine in the treatment of zoonotic cutaneous leishmaniasis (zcl) caused by l. major in iran has been studied and compared with meglumine antimoniate in a randomized open - label study. definitions of lesion cure and failure were based on both clinical and parasitological criteria two weeks after the end of treatment, and clinical recovery three months after the parasitological cure period. miltefosine was given to 32 subjects with zcl, while 31 received i m meglumine antimoniate in a dose of 20 mg sb (5) kg / daily for 14 days. of the 28 subjects who completed treatment with miltefosine, 26 were cured at 3 months corresponding to a 92.9% cure rate. there was one treatment failure (3.1%), one relapse (3.1%) and four drop - outs as a result of lack of tolerance of the drug. in the antimonial group, 25 of the 31 subjects were cured (83.3%) ; five failed (16.1%) and one (3.2%) was lost at three months of follow up. no relapses were observed in the group who received the i m meglumine antimoniate at six months follow - up after the cessation of treatment. both treatment protocols were tolerated but nausea (32.2%) and vomiting (21.5 %) were observed within the first two weeks of initiation of the oral miltefosine regimen. other gastrointestinal and musculoskeletal and total adverse reactions were not significant in the two groups. the study concluded that miltefosine was as effective as meglumine antimoniate in the treatment of zcl.13 this is a recently introduced topical immunomodulator initially approved for the treatment of genital warts. subsequently, it has been used in a wide spectrum of diseases, both inflammatory and neoplastic. in a study in peru, imiquimod cream 7.5% was used on alternate days for 20 days in a group with newly diagnosed cl. even though, there was an initial response, there was a relapse on discontinuation of treatment.14 it involves the topical application of a porphyrin precursor, aminolevulinic acid (ala) or methyl - aminolevulinate (mal), followed by irradiation with laser or intense pulsed light (ipl). since its first introduction more recently, this therapeutic modality has been utilized in non - neoplastic conditions including cutaneous leishmaniasis.15 few mechanistic studies have addressed the principles underlying the use of pdt for the treatment of cl.1618 in vitro and mechanistic studies of ala - pdt against cl did not demonstrate any parasiticidal effects on amastigotes and no differences in ala - derived ppix levels were detected between leishmania - infected and non - infected j774.2 cells.16 in contrast, in vivo topical ala - pdt performed on a murine cl model resulted in extensive tissue destruction and significant reduction of parasite load. a dramatic decrease of macrophages and these findings suggest that the parasiticidal effect of ala - pdt for cutaneous leishmaniasis is indirect and mediated through the killing of host cells rather than the direct destruction of parasites.1617 a review of six clinical studies investigating the use of pdt in 39 patients with a total of 77 lesions for the treatment of old world cl showed that pdt with the porphyrin precursors is relatively effective. however, the data is still limited and pdt can not be recommended in routine clinical practice at this point. even though it is considered a promising therapeutic modality, additional controlled trials and further investigations are needed.15 in an attempt to find an effective and alternative treatment regimen for the treatment of indian vl, where drug resistance to pentavalent antimony is high, a new approach using the combination of a single infusion of l - amb followed by a short course of oral miltefosine was tested in a randomized, non - comparative, group sequential, triangular design. a total of 181 patients participated in the study and were assigned to four treatment groups with 5mg / kg of l - amb alone (group 1 ; 45 patients) ; 5mg / kg of l - amb followed by miltefosine for 10 days (group 2 ; 46 patients) ; or 14 days (group 3 ; 45 patients) ; or 3.75 mg / kg of l - amb followed by miltefosine for 14 days (group 4 ; 45 patients). an additional 45 non - randomized patients were assigned to receive 5 mg / kg of l - amb followed by miltefosine for 7 days (group 5). all 226 subjects assigned to each of the five regimens showed an initial cure response, and a final cure rate nine months post treatment in the five groups ranged from 91 - 98%. the results of this study suggest that combination therapy with a single dose l - amb followed by 7 - 14 days of miltefosine is effective in indian kala azar.19 in a pilot study in peru, the use of imiquimod alone and in combination with parenteral meglumine antimoniate in the initial treatment of cl was compared. results of the therapy of seven patients with cl treated with topical imiquimod cream 7.5% combined with intravenous meglumine antimoniate at a dose of 20 mg / kg / day for 20 days showed a cure rate of 100% compared to 57% in the seven patients who received parenteral meglumine antimoniate alone.14 these results attest to the effectiveness of this combined therapy especially as initial treatment of cutaneous leishmaniasis in an attempt to find an effective and alternative treatment regimen for the treatment of indian vl, where drug resistance to pentavalent antimony is high, a new approach using the combination of a single infusion of l - amb followed by a short course of oral miltefosine was tested in a randomized, non - comparative, group sequential, triangular design. a total of 181 patients participated in the study and were assigned to four treatment groups with 5mg / kg of l - amb alone (group 1 ; 45 patients) ; 5mg / kg of l - amb followed by miltefosine for 10 days (group 2 ; 46 patients) ; or 14 days (group 3 ; 45 patients) ; or 3.75 mg / kg of l - amb followed by miltefosine for 14 days (group 4 ; 45 patients). an additional 45 non - randomized patients were assigned to receive 5 mg / kg of l - amb followed by miltefosine for 7 days (group 5). all 226 subjects assigned to each of the five regimens showed an initial cure response, and a final cure rate nine months post treatment in the five groups ranged from 91 - 98%. the results of this study suggest that combination therapy with a single dose l - amb followed by 7 - 14 days of miltefosine is effective in indian kala azar.19 in a pilot study in peru, the use of imiquimod alone and in combination with parenteral meglumine antimoniate in the initial treatment of cl was compared. results of the therapy of seven patients with cl treated with topical imiquimod cream 7.5% combined with intravenous meglumine antimoniate at a dose of 20 mg / kg / day for 20 days showed a cure rate of 100% compared to 57% in the seven patients who received parenteral meglumine antimoniate alone.14 these results attest to the effectiveness of this combined therapy especially as initial treatment of cutaneous leishmaniasis leishmaniasis is occasionally reported amongst transplant recipients and most cases are observed in the mediterranean area. although, this is most commonly associated with kidney transplantation (77%), it also occurs amongst patients who have undergone liver, heart, lung, pancreas and bone marrow transplantation. the most frequently observed clinical presentation in such patients is vl followed by mucosal leishmaniasis and very rarely cl. in such patients, the first line of treatment is l - amb.2022 patients with hiv / aids infection are prone to opportunistic infections such as leishmaniasis which occurs mainly in the mediterranean region, southern europe and brazil.82326 the clinical manifestations of leishmaniasis in patients with hiv are varied and include disseminated, visceral, diffuse cutaneous and post kala azar dermal leishmaniasis (pkdl). furthermore, these infections are usually resistant to treatment with pentavalent antimonials, resulting in high rate of failure and relapse. the drugs that are commonly used for hiv patients with leishmaniasis are the pentavalent antimonial compounds, amb, l - amb and miltefosine.810232527 the efficacy of the pentavalent antimonial compounds was compared with that of amb in hiv patients with leishmaniasis and showed similar initial cure rates of 66% vs. 62% respectively. however, relapse is the norm.23 apart from being an opportunistic infection in patients with aids, leishmaniasis seems to be an emerging complication in immune reconstitution inflammatory syndrome (iris) following haart therapy.28 leishmaniasis is occasionally reported amongst transplant recipients and most cases are observed in the mediterranean area. although, this is most commonly associated with kidney transplantation (77%), it also occurs amongst patients who have undergone liver, heart, lung, pancreas and bone marrow transplantation. the most frequently observed clinical presentation in such patients is vl followed by mucosal leishmaniasis and very rarely cl. in such patients, the first line of treatment is l - amb.2022 patients with hiv / aids infection are prone to opportunistic infections such as leishmaniasis which occurs mainly in the mediterranean region, southern europe and brazil.82326 the clinical manifestations of leishmaniasis in patients with hiv are varied and include disseminated, visceral, diffuse cutaneous and post kala azar dermal leishmaniasis (pkdl). furthermore, these infections are usually resistant to treatment with pentavalent antimonials, resulting in high rate of failure and relapse. the drugs that are commonly used for hiv patients with leishmaniasis are the pentavalent antimonial compounds, amb, l - amb and miltefosine.810232527 the efficacy of the pentavalent antimonial compounds was compared with that of amb in hiv patients with leishmaniasis and showed similar initial cure rates of 66% vs. 62% respectively. however, relapse is the norm.23 apart from being an opportunistic infection in patients with aids, leishmaniasis seems to be an emerging complication in immune reconstitution inflammatory syndrome (iris) following haart therapy.28 this is an anti - estrogen that has been used in the treatment and prevention of breast cancer for several years. in a recent study in which balb / c mice infected with l. amazonensis, tamoxifen was administered at a dose of 20 this resulted in a decrease in lesion size and ulcer formation, a sustained reduction in the number of parasites and extreme susceptibility. this is a promising drug that needs to be further tested.29 similar results were achieved testing the efficacy of tamoxifen in l. braziliensis - infected balb / c mice and in l. chagasi - infected hamsters.30 the pentamidine structural analogs are alkylphosphocholines used as anticancer treatment. these compounds act as membrane synthetic ether - lipid analogs and consist of alkyl chains in the lipid portions. the most promising of these are miltefosine (hexadecyl phosphocholine), eldofisine [et-18-och (3) ], ilmofosoine (bm41.440) and perofisine.31 based on the proven efficacy of pentamidine and related dications against protozoal infections, 18 structural analogs of pentamidine were evaluated for in vitro anti - leishmanial activity of l. major and l. tropica. results showed that the reversed amidine compounds were more active than the furan analogs against both leishmania species. db 745 and db 746 demonstrated the highest activity against l major, while db 745 was more effective against l. tropica. both compounds, however, exhibited 50% inhibitory concentrations (ic 50) below 1 nm for l. major. of the 10 reversed amidine compounds, nine showed inhibitory growth effect on amastigote axenic model at a concentration below 1 nm. these studies show that dicationic compounds are potential new agents with less toxicity than the parent drug for the treatment of cl, but further studies are needed.32 other pentamidine analogs tested for activity against leishmania organisms are parfuramidine, eflornithine, perifosine, edelfosine, ilmosfosine and sitamaquine.313336 parfuramidine is a novel diamidine already in phase iii clinical trial for early stage disease of human african trypanosomiasis (hat).33 perifosine, another novel alkylphospholipid, which recently entered phase ii clinical trials, demonstrated high in vitro activity against all tested strains of leishamania including l. amazonensis.34 both edelfsoine and perifosine demonstrated in vivo activity against l. amazonensis as shown in a study in which edelfosine and perifosine were given to balb / c mice in oral doses of 1 and 2.5 mg / kg / day during 28 days and 5 mg / kg/ day during 14 days, starting at two weeks after the first treatment scheme.34 an assay comparing miltefosine at the standard dose of 20 mg / kg / day during 28 days to in vivo treatment with perifosine at a dose of 5 mg / kg / day for 14 days demonstrated higher in vivo activity of perifosine than miltefosine against l. amazonensis. these findings show promise for a new treatment group in cutaneous leishmaniasis caused by l. amazonensis.34 another drug, sitamaquine (wr 6026), an 8- aminoquinoline, currently being tested and in phase ii clinical trials has also shown promise as an effective oral agent in a dose of 1 mg / kg / day for 2 weeks for visceral leishmaniasis. further studies are needed.36 the present focus is on identifying newer therapeutic targets in the parasite such as dna topoismerases.31 undoubtedly, in the coming years, a group of new medications safe and effective for the treatment of leishmaniasis will be found. recent work on sand - fly saliva has identified maxadilan, a vasoactive peptide that acts on the pac-1 receptor.37 this results in vasodilation and consequently enhances infectivity. possibilities of identifying medications that affect the pac-1 receptor can help in the treatment of leishmaniasis, as well as in the production of a vaccine that will protect against this infection. undoubtedly, there are other aspects of the management of leishmaniasis in endemic areas. once the infection is present, vaccines and vector control are paramount in addition to therapeutic measures, but these are beyond the scope of this review. this is an anti - estrogen that has been used in the treatment and prevention of breast cancer for several years. in a recent study in which balb / c mice infected with l. amazonensis, tamoxifen was administered at a dose of 20 this resulted in a decrease in lesion size and ulcer formation, a sustained reduction in the number of parasites and extreme susceptibility. this is a promising drug that needs to be further tested.29 similar results were achieved testing the efficacy of tamoxifen in l. braziliensis - infected balb / c mice and in l. chagasi - infected hamsters.30 the pentamidine structural analogs are alkylphosphocholines used as anticancer treatment. they have recently been found to be very effective as oral treatment for leishmaniasis. these compounds act as membrane synthetic ether - lipid analogs and consist of alkyl chains in the lipid portions. the most promising of these are miltefosine (hexadecyl phosphocholine), eldofisine [et-18-och (3) ], ilmofosoine (bm41.440) and perofisine.31 based on the proven efficacy of pentamidine and related dications against protozoal infections, 18 structural analogs of pentamidine were evaluated for in vitro anti - leishmanial activity of l. major and l. tropica. results showed that the reversed amidine compounds were more active than the furan analogs against both leishmania species. db 745 and db 746 demonstrated the highest activity against l major, while db 745 was more effective against l. tropica. both compounds, however, exhibited 50% inhibitory concentrations (ic 50) below 1 nm for l. major. of the 10 reversed amidine compounds, nine showed inhibitory growth effect on amastigote axenic model at a concentration below 1 nm. these studies show that dicationic compounds are potential new agents with less toxicity than the parent drug for the treatment of cl, but further studies are needed.32 other pentamidine analogs tested for activity against leishmania organisms are parfuramidine, eflornithine, perifosine, edelfosine, ilmosfosine and sitamaquine.313336 parfuramidine is a novel diamidine already in phase iii clinical trial for early stage disease of human african trypanosomiasis (hat).33 perifosine, another novel alkylphospholipid, which recently entered phase ii clinical trials, demonstrated high in vitro activity against all tested strains of leishamania including l. amazonensis.34 both edelfsoine and perifosine demonstrated in vivo activity against l. amazonensis as shown in a study in which edelfosine and perifosine were given to balb / c mice in oral doses of 1 and 2.5 mg / kg / day during 28 days and 5 mg / kg/ day during 14 days, starting at two weeks after the first treatment scheme.34 an assay comparing miltefosine at the standard dose of 20 mg / kg / day during 28 days to in vivo treatment with perifosine at a dose of 5 mg / kg / day for 14 days demonstrated higher in vivo activity of perifosine than miltefosine against l. amazonensis. these findings show promise for a new treatment group in cutaneous leishmaniasis caused by l. amazonensis.34 another drug, sitamaquine (wr 6026), an 8- aminoquinoline, currently being tested and in phase ii clinical trials has also shown promise as an effective oral agent in a dose of 1 mg / kg / day for 2 weeks for visceral leishmaniasis. further studies are needed.36 the present focus is on identifying newer therapeutic targets in the parasite such as dna topoismerases.31 undoubtedly, in the coming years, a group of new medications safe and effective for the treatment of leishmaniasis will be found. recent work on sand - fly saliva has identified maxadilan, a vasoactive peptide that acts on the pac-1 receptor.37 this results in vasodilation and consequently enhances infectivity. possibilities of identifying medications that affect the pac-1 receptor can help in the treatment of leishmaniasis, as well as in the production of a vaccine that will protect against this infection. undoubtedly, there are other aspects of the management of leishmaniasis in endemic areas. once the infection is present, vaccines and vector control are paramount in addition to therapeutic measures, but these are beyond the scope of this review. at present, the pentavalent antimonial compounds remain the mainstay of the treatment for cutaneous leishmaniasis. however, because of the high toxicity associated with this group and the recent emergence of drug resistant strains, alternative therapeutic options to be considered are the pentamidines and liposomal amphotericin b. the most effective alternative to antimonials are combination therapies using l - amb and miltefosine. photodynamic therapy, imiquimod, and tamoxifen show promise for the near future. in immunocompromised individuals, miltefosine alone or in combination with the antimonials and l - amb have proved to be effective. amphotericin b and the pentavalent antimonials are relatively cheaper but the efficacy and toxicity of these outweigh the costs of the newer agents. the choice of anti - leishmanial therapy should be based on the geographic location, availability of the drug, host immune status and expertise of the treating physician. these new treatments are not expected to be available at the phc but rather in specialized centers / hospitals.	background : cutaneous leishmaniasis (cl) is still a major health problem in many countries including saudi arabia. patients with cl are seen, not only by dermatologists, but also by pediatricians and community physicians. knowledge of available treatment options is essential.design:a literature review utilizing pubmed and cochrane evidence - based library was undertaken in the last five years.results:several medications and therapeutic modalities are currently in use, though the gold standard remains systemic antimonials. drug resistance and serious side effects preclude the use of available medications. newer therapies like liposomal amphotericin b, miltefosine and pentamidine are being used ; while it is hoped that other drugs like imiquimod, tamoxifen, pdt and pentamidine structural analogs being tested would offer better efficacy, easier administration and lower toxicity.conclusion:after decades of little advance in the treatment of leishmaniasis, there are now several options with newer compounds and combinations of these.
aerobic fitness is essential for soccer athletes to perform at an optimal level (15, 17). in addition to aerobic fitness, repeated sprint performance is also a critical component of game performance in soccer. the ability to reproduce sprints without significant reductions in sprint speed is known as repeated sprint ability (rsa). athletic performance at a high level requires soccer athletes to train both their oxidative phosphorylation (aerobic) system and their phosphocreatine - adenosine triphosphate (pcr - atp) (anaerobic) system (7, 16). while the pcr - atp system is the primary system to fuel athletes during the anaerobic (i.e., burst of sprints lasting less than 6 seconds) portions of a game, the aerobic system also plays an important role to assist with pcr replenishment (7, 9, 21). the relationship between the aerobic and anaerobic variables on an athlete s ability to reproduce sprints is debatable and the current research is discordant due to the varying forms of a repeated sprint test (1, 2, 5, 16). a better understanding about the relationship between aerobic fitness and brief anaerobic work in the form of repeated sprints that are less than 40 meters is needed to improve strength and conditioning recommendations for soccer athletes. prior research has yet to show a relationship between aerobic fitness (e.g., vo2max) and rsa when the repeated sprint distances were less than 40 meters and when the work - to - rest ratios are 1:4 to 1:5 (6, 21). a study by aziz. (1) concluded that aerobic fitness measured via vo2max was not associated with rsa sprint performance in young elite soccer athletes. the rsa test implemented used a rsa running test that consisted of six, 20 meter sprints with a 20 second recovery between each sprint (~1:5 work - to - rest ratio). another rsa study increased the distance by 10 meters with the same recovery time and also suggested that rsa is not related to aerobic fitness (14). (14) was that vo2max was not objectively measured, but estimated using a shuttle run. contrary to studies suggesting no relationship between vo2max and rsa, some research has provided evidence that vo2max is related to rsa (5, 12). for example, results from bishop and spencer (5) suggest that aerobic fitness, in addition to anaerobic power, is related to rsa performance in well - trained adult athletes when using a wind - braked cycle ergometer protocol consisting of 5 repetitions, 6 seconds of work with a 24 second recovery between repetitions (1:4 work - to - rest ratio) (5) 12) implemented a rsa test consisting of 6, 40-meter sprints with 20 seconds of recovery between each sprint and found that vo2max is associated with rsa. a potential reason for the discrepancies between these studies could be due to the different sprint distances (2030 meters versus 40 meters), modes of exercise (i.e., running compared to cycling) employed for the rsa tests and even possibly the age and level of athletes (i.e., young versus adult and elite versus vs. non - elite) tested. to better assess the relationship between vo2max and rsa in soccer athletes, a running sprint test should be utilized with shorter sprint distances than 40 meters. it is reasonable to suggest that soccer athletes frequently engage in sprint distances of less than 40 meters and it is imperative to assess if aerobic fitness is still related to a soccer athletes rsa when shorter sprint distances are utilized. the potential relationship, or lack thereof, between sprint times and vo2max could alter optimal training recommendations. in addition to vo2max and sprint times, the previously aforementioned studies also assessed factors such as total sprint time (i.e., sum of all repeated sprint times in the protocol) and average sprint time (1, 2, 5, 10, 12, 16). it has been established that utilizing average sprint time is an effective measure to assess rsa performance (1, 13, 14, 21). rsa can be used to calculate an athletes fatigue using a fatigue index (i.e., percent reduction of sprint time from the fastest sprint) in relation to the aerobic capacity throughout a rsa test (21). while rate of fatigue is an intriguing calculation and assessment, it has been reported to be less reliable, relative to average sprint time, when analyzing rsa performance (13). however, a better understanding is needed to assess the relationship between aerobic capacity and rate of fatigue when employing different rsa protocols, primarily with shorter sprint distance and more sprint repetitions. to justify changes in rsa, longer sprint distances, quicker recovery times and additional sprints should simulate a more difficult test in an attempt to replicate a more realistic soccer - conditioning test. therefore, the purpose of the study was to directly measure division i collegiate soccer athletes vo2max and then assess its relationship to rsa test performance, rate of fatigue and the age of the athletes when a more challenging sprint test is employed. based on previous research, it is hypothesized that vo2max will be related to rate of fatigue and age, but not rsa performance since each sprint distance was less than 40 meters. a total of 20 (n = 10 females, n = 10 males) division i soccer athletes volunteered to participate in the study. all athletes were cleared by the team physician and had no contraindications to physical activity or exercises. prior to participation in the study, research personnel explained the vo2max protocol (bruce protocol) and the procedures for the rsa test. after explaining the research protocol, athletes read and signed an informed consent form. the testing protocol for vo2max included a validated, graded maximal treadmill exercise test (bruce protocol) (8) and the rsa test which consisted of 10 total 30-meter sprints, one sprint every 30 seconds. since the rsa testing protocols in the literature vary from study to study, the rsa test employed in the current study required four more sprints (10, as opposed to 6) and a sprint distance of 30 meters. the combination of a 30-meter sprint distance, quicker recovery time and additional sprints simulated a more physiologically taxing soccer - specific conditioning test. upon arrival to the human performance laboratory, trained research personnel measured each athlete s height and weight using a stadiometer and balance beam scale (detecto, webb city, mo usa), respectively. after anthropometric measurements were recorded, athletes were fitted with a heart rate monitor (polar, kempele, finland) and a full vo2 mask and head strap for the vo2max test (hans rudolph, inc, kansas, usa). following initial measures, athletes completed a maximum of a ten - minute warm - up jog at a self - selected pace. upon completion of the warm up, the vo2 mask and head strap was appropriately secured to each athlete and was then connected to a calibrated metabolic cart with his or her heart rate monitor synced to the cart. oxygen consumption was recorded using a gas and flow calibrated metabolic cart (pravo medics, truemax 2400) and vo2 was recorded as relative vo2 in ml. each subject s respiratory exchange ratio (rer) was monitored to ensure maximum effort was exerted. vo2max was achieved if they discontinued to run and their rer was 1.15. after completing the vo2max test, athletes performed a cool - down that consisted of a self - selected walking pace to reduce any risk of injury or syncope episodes. athletes were advised to rest for a minimum of 2448 hours after the vo2max test. to avoid any influence from vo2max testing, research personnel scheduled all rsa testing a week after subjects completed their vo2max test. for the rsa test, each athlete completed a 10-minute active warm - up that was specific to the linear sprinting movement and distance that was encountered during the test including a static stretching session in which the athletes had the freedom to stretch any muscles they needed. following the warm - up, all sprint times were recorded via stopwatch by the one trained researcher to minimize tester error. percent heart rate was recorded to assess maximal efforts throughout the rsa test. for the rsa test, once the first 30-meter sprint was completed, the athlete rested and then began the next sprint 30 seconds after their previous sprint started. therefore, the actual rest time between each sprint varied based on the sprint time of each athlete. after deceleration, the work - to - rest ratio was approximately 1:4 (~ 6 seconds of work, 24 seconds of rest). upon completion of the rsa test, athletes participated in an active cool - down for five minutes to allow their heart rate to safely return to resting levels followed by a five - minute static stretch. fatigue index (fi) was calculated upon completion of the study to assess rate of fatigue as a percent. to calculate rate of fatigue for each athlete, descriptive statistics using mean and standard deviation were calculated for all physical characteristics (e.g., age, height, and weight) and performance (e.g., vo2max, average sprint time, percent heart rate maximum, and fatigue index). differences between males and females were analyzed using an independent samples t - test. a single, three - time point (1st, 5th, and 10th sprint) repeated measures analysis of variance (anova) was utilized to assess any differences in percent of maximal heart rate that athletes exerted. post hoc analyses were conducted using paired samples t - test and the benjamini and hochberg false discovery rate correction for multiple comparisons (3). males and females percent maximal heart rate was not significantly different ; therefore, gender was excluded from the anova model. lastly, multiple correlation analyses were utilized to assess if any relationships exists between vo2max, age, average rsa sprint times, and rate of fatigue. gender was excluded from all correlation models due to the small number of participants in each group (n = 10). all statistics were analyzed using ibm spss 21.0 (version 21.0, ibm inc, armonk, ny). a total of 20 (n = 10 females, n = 10 males) division i soccer athletes volunteered to participate in the study. all athletes were cleared by the team physician and had no contraindications to physical activity or exercises. prior to participation in the study, research personnel explained the vo2max protocol (bruce protocol) and the procedures for the rsa test. after explaining the research protocol, athletes read and signed an informed consent form. the testing protocol for vo2max included a validated, graded maximal treadmill exercise test (bruce protocol) (8) and the rsa test which consisted of 10 total 30-meter sprints, one sprint every 30 seconds. since the rsa testing protocols in the literature vary from study to study, the rsa test employed in the current study required four more sprints (10, as opposed to 6) and a sprint distance of 30 meters. the combination of a 30-meter sprint distance, quicker recovery time and additional sprints simulated a more physiologically taxing soccer - specific conditioning test. upon arrival to the human performance laboratory, trained research personnel measured each athlete s height and weight using a stadiometer and balance beam scale (detecto, webb city, mo usa), respectively. after anthropometric measurements were recorded, athletes were fitted with a heart rate monitor (polar, kempele, finland) and a full vo2 mask and head strap for the vo2max test (hans rudolph, inc, kansas, usa). following initial measures, athletes completed a maximum of a ten - minute warm - up jog at a self - selected pace. upon completion of the warm up, the vo2 mask and head strap was appropriately secured to each athlete and was then connected to a calibrated metabolic cart with his or her heart rate monitor synced to the cart. oxygen consumption was recorded using a gas and flow calibrated metabolic cart (pravo medics, truemax 2400) and vo2 was recorded as relative vo2 in ml. each subject s respiratory exchange ratio (rer) was monitored to ensure maximum effort was exerted. vo2max was achieved if they discontinued to run and their rer was 1.15. after completing the vo2max test, athletes performed a cool - down that consisted of a self - selected walking pace to reduce any risk of injury or syncope episodes. athletes were advised to rest for a minimum of 2448 hours after the vo2max test. to avoid any influence from vo2max testing, research personnel scheduled all rsa testing a week after subjects completed their vo2max test. for the rsa test, each athlete completed a 10-minute active warm - up that was specific to the linear sprinting movement and distance that was encountered during the test including a static stretching session in which the athletes had the freedom to stretch any muscles they needed. following the warm - up, all sprint times were recorded via stopwatch by the one trained researcher to minimize tester error. percent heart rate was recorded to assess maximal efforts throughout the rsa test. for the rsa test, once the first 30-meter sprint was completed, the athlete rested and then began the next sprint 30 seconds after their previous sprint started. therefore, the actual rest time between each sprint varied based on the sprint time of each athlete. after deceleration, the work - to - rest ratio was approximately 1:4 (~ 6 seconds of work, 24 seconds of rest). upon completion of the rsa test, athletes participated in an active cool - down for five minutes to allow their heart rate to safely return to resting levels followed by a five - minute static stretch. fatigue index (fi) was calculated upon completion of the study to assess rate of fatigue as a percent. to calculate rate of fatigue for each athlete, descriptive statistics using mean and standard deviation were calculated for all physical characteristics (e.g., age, height, and weight) and performance (e.g., vo2max, average sprint time, percent heart rate maximum, and fatigue index). differences between males and females were analyzed using an independent samples t - test. a single, three - time point (1st, 5th, and 10th sprint) repeated measures analysis of variance (anova) was utilized to assess any differences in percent of maximal heart rate that athletes exerted. post hoc analyses were conducted using paired samples t - test and the benjamini and hochberg false discovery rate correction for multiple comparisons (3). males and females percent maximal heart rate was not significantly different ; therefore, gender was excluded from the anova model. lastly, multiple correlation analyses were utilized to assess if any relationships exists between vo2max, age, average rsa sprint times, and rate of fatigue. gender was excluded from all correlation models due to the small number of participants in each group (n = 10). all statistics were analyzed using ibm spss 21.0 (version 21.0, ibm inc, armonk, ny). males weighed significantly more, were taller, older, faster and had greater aerobic capacities than female athletes (p < 0.046, table 1). the correlation analysis that tested the relationship between vo2max and rsa revealed there were significant (p < 0.001, table 2) negative correlations between vo2max and all 10 repeated sprint times. after averaging all ten rsa sprint times, there was a significant negative relationship between vo2max and average sprint time (r =.767, p < 0.001). there was no significant relationship between vo2max and athlete s rate of fatigue calculated using a fatigue index (r =.333, p = 0.15). there was main effect of condition for percent of maximum heart rate (p = 0.001). athlete s percent of maximum heart rate increased from the 1 (73%) to the 5 (87%) sprint and then increased again after the 10 sprint (93.5%). independent samples t - test revealed that athletes, regardless of gender, contributed a similar percent of their maximum effort (assessed via heart rate) throughout the rsa test (p 0.127 for all). there was a significant positive relationship between vo2max and age of the athletes, (r =.451, p =.046). contrary to the hypothesis, the negative correlations found suggest that vo2max or aerobic capacity does play a significant role in repeated sprint performance in that more aerobically fit athletes are faster than their less aerobically fit peers. these results coincide with previous research that suggests vo2max is related to improved rsa test performance (20). tomlin and wenger (20), postulated, and the current results support, that athletes with greater aerobic capacity may have superior power recovery and can perform well during repeated high intensity work. additionally, the current results are contrary to previous research suggesting aerobic fitness does not significantly influence sprint performance during an rsa test of less than 40 meters because the anaerobic energy needed to complete a five second sprint is less likely to be influenced by oxygen uptake (1, 7). while the energy demands of a single sprint may not be influenced by aerobic fitness, it appears that faster repeated sprint times and perhaps rsa may actually be associated with greater levels of aerobic fitness, even if the sprint distance is less than 40 meters. in addition to vo2max and rsa times, rate of fatigue is an important factor to assess when examining a soccer athlete s conditioning level or anaerobic capacity. in the current study while anaerobic capacity was not measured via a wingate test, the small percentage in which athletes fatigued suggests the athletes were well trained anaerobically. the low rate of fatigue in addition to the aerobic capacity results highlight the importance for strength and conditioning coaches to emphasize training both aerobic fitness and anaerobic capacity. research strongly suggests the use of 1:1 or 2:1 work - to - rest ratios to maximize training for the improvement of both of aerobic and anaerobic pathways (11, 18, 19). a vo2max of 60 mlkgmin or greater has been used as a physiological indicator for researchers to establish a minimum fitness level for men s professional soccer athletes (17). on average, male athletes in the current study had an averaged vo2max of 66.3 mlkgmin, which is well above the aforementioned minimum for professional or elite status. moreover, these results also suggests that younger college athletes should begin training and conditioning aimed to improve their vo2max. there was a positive relationship between the athlete s age and vo2max suggesting that older college athletes in the study had a greater vo2max and a plausible explanation for the relationship may be due to the fact the older athletes have trained more and with greater intensities throughout their college career. although training age (i.e., number of years training) was not reported in the current study, it could be advantageous for younger soccer athletes to focus their training on methods to improve aerobic (vo2max) and anaerobic capacity to potentially perform better on an rsa test. while the results reveal a strong negative relationship between vo2max and rsa sprint performance, it is not without limitations. anaerobic capacity and rate of fatigue were not measured using a validated anaerobic capacity test such as the wingate test or a running anaerobic sprint test (22). using a validated running anaerobic capacity test would provide useful information regarding peak and minimum power outputs. using peak and minimum outputs may provide a better calculation of rate of fatigue and anaerobic capacity as opposed to using fastest and slowest sprint times. understanding power outputs and power recovery, in addition to vo2max, may allow for better strength and conditioning programming to improve rate of fatigue and rsa performance (20). another limitation could be that factors other than vo2max could be associated with rsa performance (i.e., nutrition, sleep, and recovery) and were not assessed in the current study. although there are other physiological factors to consider when testing athletes rsa, the purpose of the study was to assess if vo2max was associated with rsa performance when sprint distances of less than 40 meters were employed. shorter sprint distances with ~25 seconds of recovery are believed to not be influenced by aerobic metabolism s assistance with pcr repletion (9) and the current results suggest otherwise. division i soccer athletes that are more aerobically fit (i.e., greater vo2max) appear to be faster on average and during every sprint than their less aerobically fit peers when participating in a 30-meter repeated sprint test. training to enhance a soccer athlete s aerobic fitness is highly recommended.	research is inconclusive regarding the association between aerobic fitness (objectively measured vo2max) and repeated sprint performance when the sprints are less than 40 meters. soccer athletes must be able to repeat sprints without significant decreases in speed and strength and conditioning coaches need to better understand if aerobic fitness is related to repeated sprint ability (rsa). twenty (10 male, 10 female) division i soccer athletes first completed a graded maximal treadmill test to measure vo2max. then on a separate day, athletes completed the rsa test. the rsa test consisted of 10, 30-meter sprints which athletes repeated every 30 seconds. there were significant negative correlations (r 0.69, p < 0.001) between vo2max and all 10-sprint times and average sprint time. more aerobically fit division i soccer athletes were faster at all time points during the rsa test. aerobic fitness is associated with faster sprint times during a more anaerobic rsa test when sprint distances are less than 40 meters.
for many years, the diagnosis of lung metastasis was considered the end stage of the disease course in patients with malignant neoplasms. initially, the treatment proposed consisted of chemotherapy and hormone therapy, and unsatisfactory responses were seen in more than 70% of cases in 1882, weinlechner incidentally performed the first lung metastasis resection during the resection of a primary tumor, a procedure in which the metastasis was removed en bloc with the surgical specimen. () since then, various other cases of metastasectomy have been reported, many with positive results. in 1951, ehrenhaft started selecting candidates for lung metastasis resection, () and, subsequently, it was possible to establish well - defined criteria. surgical treatment evolved over the 20th century because of greater knowledge about tumors that gave rise to lung metastases only. surgical treatment of lung metastases has been given a boost in the last 15 years, as has been shown in the literature, and some of the major prognostic factors for this condition, such as disease - free interval and type of resection performed, have been identified,. (,) however, in comparison with major centers worldwide, there have been few studies of brazilian patients and this treatment modality in brazil. therefore, the objective of the present study was to evaluate the results of surgical treatment of lung metastases at a university hospital in the city of campinas, brazil. this was a retrospective study using surgical records of the department of thoracic surgery of the state university at campinas hospital de clnicas, located in the city of campinas, brazil. we reviewed all records of resection of lung lesions performed in patients with a previous diagnosis of primary cancer in other organs, as well as all records of complete resection of all lesions and pathological confirmation of metastatic disease, between january 1, 1997 and december 31, 2011. we included all patients with lung lesions who were selected for surgery on the basis of the thomford et. al. criteria for surgical treatment of metastatic lung lesions, () namely : a) complete resection of all metastatic disease should be possible ; b) the patient should have sufficient pulmonary function to withstand the risk of surgery and survive the postoperative period ; c) the primary tumor should be (or can be) controlled ; d) there should be no evidence of extrapulmonary metastases, except for colon tumors ; and e) there should be no effective treatment other than surgery. pulmonary function was assessed on the basis of arterial blood gas analysis results and pulmonary function test results. if the pulmonary function test was not technically possible, the six - minute walk test, which has been proven to be well tolerated and safe in the assessment of patient functional capacity, was used. () in addition, the records were reviewed for the postoperative presence of systemic inflammatory response syndrome (sirs), which was defined by the presence of at least two of the following parameters, on the same day, within three days after surgery : an axillary temperature above 38c ; an rr > 24 breaths / min ; an hr > 90 bpm ; and a leukocyte count greater than 10,000 cells / mm or less than 5,000 cells / mm. overall survival was defined as the date of first surgery to the last follow - up visit or to death (from any cause). to determine disease - free intervals, we used the dates of primary cancer treatment and the date of referral for thoracic surgery for treatment of lung metastasis. we designed a data collection form that was used by two trained physicians to obtain the patient medical record information needed to meet the objectives of the present study. this form contained the following fields : patient initials ; medical record number ; gender ; race ; date of birth ; date of surgery of the primary tumor ; date of referral for thoracic surgery ; symptoms at diagnosis of metastasis (dyspnea, cough, hemoptysis, and chest pain) ; smoking ; anatomical site of the primary tumor (breast, head, neck, colorectum, kidney, musculoskeletal system, gonads, etc.) ; presence and type of comorbidity ; history of resection for liver metastasis ; neoadjuvant or adjuvant therapy to treatment of lung metastasis ; number of lung resections ; number and location of nodules found intraoperatively ; date of resection of the metastasis ; type of resection performed (segmentectomy, wedge resection, nodule resection, lobectomy, bilobectomy, and pneumonectomy) ; use of a stapler ; extubation in the operating room ; number of chest tubes and side of placement ; unilateral or bilateral approach ; surgical access (thoracotomy, bilateral sequential thoracotomy, sternotomy, and video - assisted surgery) ; need for blood transfusion ; postoperative recovery in the icu ; development of sirs ; length of hospital stay ; presence and type of complication within 30 days after surgery ; postoperative day of onset of the complication ; current status of the patient (alive / dead) ; date of the last follow - up visit to our facility ; and whether follow - up was conducted at another facility. data on postoperative complications, defined as any type of event experienced by patients within 30 days after surgery, were extracted from patient medical records. these data were grouped by major system or pathology involved, listed by frequency, and stratified as a variable in the overall survival curve. the present study was approved by the research ethics committee of the state university at campinas school of medical sciences (protocol no. for categorical variables, we conducted an exploratory descriptive analysis and, subsequently, we used pearson 's, spearman 's, and fisher 's tests, whereas, for continuous variables, we used the t - test (two groups) or the kruskal - wallis test (more than two groups). in cases with missing data, the valid percent of variables was calculated. the kaplan - meier method was used for survival analyses, and the log - rank test was used for survival comparisons. cox regression analysis (by wald 's backward stepwise method) was performed to identify the predictive variables over time. for univariate and multivariate analyses, the level of significance was set at p 24 ciclos / min ; fc > 90 bpm ; leucograma maior que 10.000 clulas / mm ou menor que 5.000 clulas / mm. definiu - se a sobrevida global a partir da data da primeira cirurgia at a data da ltima consulta ou bito (por qualquer causa). para o clculo do intervalo livre de doena, foram utilizadas as datas do tratamento da neoplasia primria e a data do encaminhamento cirurgia torcica para o tratamento da metstase pulmonar. foi elaborada uma ficha de coleta de dados utilizada por dois mdicos treinados para a obteno das informaes dos pronturios dos pacientes necessrias para os objetivos definidos do estudo. essa ficha era composta pelos seguintes campos : iniciais do paciente ; nmero do pronturio ; sexo ; raa ; data de nascimento ; data da cirurgia do tumor primrio ; data do encaminhamento para a cirurgia torcica ; sintomas ao diagnstico da metstase (dispneia, tosse, hemoptise e dor torcica) ; tabagismo ; stio anatmico do tumor primrio (mama, cabea, pescoo, colorretal, renal, musculoesqueltico, gnadas, etc.) ; presena e tipo de comorbidade associada ; resseco de metstase heptica prvia ; terapia neoadjuvante ou adjuvante ao tratamento da metstase pulmonar ; nmero de resseces pulmonares ; nmero e localizao de ndulos encontrados no intraoperatrio ; data da resseco da metstase ; tipo de resseco utilizada (segmentectomia, cunha, nodulectomia, lobectomia, bilobectomia e pneumonectomia) ; uso de grampeador ; extubao em sala cirrgica ; nmero e lateralidade de drenos torcicos ; abordagem uni ou bilateral ; via de acesso (toracotomia, bitoracotomia sequencial, esternotomia e videocirurgia) ; necessidade de transfuso sangunea ; recuperao ps - operatria em uti ; desenvolvimento de sirs ; tempo de internao hospitalar ; presena e tipo de complicao at 30 dias da cirurgia ; dia do ps - operatrio em que iniciou a complicao ; situao atual do paciente (vivo / bito) ; data da ltima consulta no servio ; e se fez seguimento em outro servio. as complicaes ps - operatrias foram extradas dos pronturios dos pacientes, definidas como todo e qualquer tipo de evento que tenha ocorrido com o paciente no perodo de 30 dias aps o ato cirrgico. agrupamos esses dados de acordo com os principais sistemas ou patologias envolvidos, os listamos por frequncia e os estratificamos como uma varivel na curva de sobrevida global. o presente estudo foi aprovado pelo comit de tica em pesquisa da faculdade de cincias mdicas da universidade estadual de campinas sob o protocolo no. 001/2013. aplicou - se a anlise descritiva exploratria e, posteriormente, os testes de pearson, spearman e fisher, no caso de variveis categricas, enquanto no caso de variveis contnuas, foram utilizados o teste t (dois grupos) ou o teste de kruskal - wallis (mais de dois grupos). para as anlises de sobrevida, aplicou - se o mtodo de kaplan - meier e, para seus cotejamentos, o teste de log - rank. a regresso de cox foi aplicada na busca de variveis preditivas no decorrer do tempo, com o mtodo de wald (backward stepwise). para as anlises univariada e multivariada, o nvel de significncia considerado foi de, respectivamente, p < 0,10 e p < 0,05. as anlises foram realizadas atravs do programa estatstico statistical package for the social sciences, verso 14.0 (spss inc., durante o perodo estudado, 119 pacientes foram submetidos a um total de 154 procedimentos operatrios. desses pacientes, 68 (57,1%) eram do sexo masculino, com uma mediana de idade de 52 anos (variao, 15 - 75 anos), e 108 (90,8%) eram brancos. os principais stios de origem do tumor primrio encontrados foram colorretal, em 47,9% dos pacientes ; musculoesqueltico, em 21,8% ; cabea e pescoo, em 7,5% ; e gnadas, em 5,9%. as comorbidades associadas eram presentes em 63 pacientes (52,9%), sendo as mais frequentes a hipertenso arterial sistmica, em 69,8% ; diabetes, em 19,0% ; asma / dpoc, em 7,9% ; dislipidemia, em 7,9% ; hipertireoidismo, em 6,3% ; e cardiopatia, em 6,3%. o nmero de procedimentos operatrios realizados nos pacientes foi de um, dois, trs e quatro, respectivamente, em 91 pacientes (76,5%), 24 (20,2%) ; 3 (2,5%) e 1 (0,8%). foram submetidos resseco de metstase heptica antes do diagnstico da metstase pulmonar 10 pacientes (8,4%). da mesma forma, 85 pacientes (71,4%) realizaram quimioterapia e / ou radioterapia antes da resseco da metstase pulmonar. aps a resseco, 88 pacientes (75,9%) realizaram quimioterapia adjuvante (tabela 1). a taxa de recidiva de metstase pulmonar foi de 19,3%. a mediana do intervalo de tempo livre de doena foi de 23 meses (variao, 1 - 172 meses). a mdia de dias de internao hospitalar foi de 8 dias (variao, 3 - 40 dias) em relao aos 154 procedimentos operatrios. a localizao preferencial das leses ressecadas foi no lobo inferior esquerdo (24,8%), seguida no lobo superior esquerdo (24,4%) e no lobo inferior direito (21,5%). com relao ao ato operatrio, foram abordadas leses bilaterais no mesmo tempo cirrgico em 25 procedimentos operatrios (16,3%), enquanto somente um dos pulmes foi abordado em 129 procedimentos (83,7%). desses 129 procedimentos cirrgicos, 20 (em 10 pacientes) foram realizados em um intervalo mdio de 2 meses a fim de tratar doena bilateral, que estava presente em 35 pacientes (29,4%) ao diagnstico da metstase pulmonar. as vias de acesso utilizadas mais comuns foram toracotomia, em 78,6% dos casos, seguida por esternotomia, em 9,1% ; bitoracotomia sequencial, em 8,5% ; e videocirurgia, em 3,8%. foi considerada somente como um ato operatrio. os tipos de resseces foram resseco em cunha ou segmentectomia, em 51,3% dos casos ; nodulectomia, em 29,9% ; lobectomia ou bilobectomia, em 15,6% ; e pneumonectomia, em 3,2%. de um total de 154 atos operatrios, utilizou - se o grampeador linear para auxlio em 10 procedimentos (6,5%), os pacientes no foram extubados na prpria sala cirrgica, e 12 (7,8%) no fizeram recuperao do ps - operatrio imediato em ambiente de uti. aps os atos operatrios, 28 pacientes (18,2%) evoluram com sirs, e 9 desses cursaram com algum tipo de complicao perioperatria. no perodo perioperatrio, 34 pacientes (22,1%) tiveram algum tipo de complicao, sendo as mais comuns os quadros pulmonares (pneumonia, insuficincia respiratria, ventilao mecnica prolongada, cogulo retido, edema pulmonar, fistula area persistente) em 25 (16,2%) e quadros extrapulmonares (insuficincia cardaca, arritmia, infeco de parede / trato urinrio e tromboembolismo) em 12 (7,8%). a mortalidade perioperatria foi de 3 pacientes (1,9%) devida a arritmia irreversvel no intraoperatrio em 1 e a complicaes pulmonares em 2 (tabela 2). tabela 2 caractersticas dos 154 atos operatrios realizados durante o perodo do estudo. dos 119 pacientes estudados, 64 (53,8%) mantiveram seguimento ambulatorial, enquanto 22 (18,5%) perderam contato com o servio por mais de 1 ano, e 33 (27,7%) foram a bito. a tabela 3 apresenta as taxas de sobrevida global dos pacientes em 12, 36, 60 e 120 meses, equivalentes a 96%, 77%, 56% e 39% respectivamente. a sobrevida global para os grupos que realizaram ou no quimioterapia ou radioterapia antes (48% e 72%) e depois (51% e 72%) da cirurgia apresentou uma diferena significativa (p = 0,02) a favor daqueles pacientes que no as realizaram, porm sem confirmao na anlise multivariada. ao se cotejar os pacientes sem e com complicaes nos primeiros 30 dias aps a cirurgia (67% vs. 24% ; p < 0,0001), a anlise de cox confirmou a piora no prognstico (hazard ratio = 1,81 ; ic95% : 1,09 - 3,06 ; p = 0,02 ; figura 1). figura 1 em a, sobrevida global nos grupos de pacientes submetidos ou no a quimioterapia / radioterapia (qt / rt) antes da cirurgia (n = 119). em b, sobrevida global nos grupos de pacientes submetidos ou no a qt / rt depois da cirurgia (n = 116). em c, sobrevida global nos grupos de pacientes que apresentaram ou no complicaes nos em at 30 dias ps - operatrios (n = 119). estratificando a sobrevida pelo stio anatmico do tumor primrio encontramos uma sobrevida global em 60 meses de 68% no carcinoma colorretal, de 26% no carcinoma musculoesqueltico e de 56% nos demais grupos (menos frequentes que os anteriores ; figura 2). figura 2 sobrevida global nos pacientes em funo do tipo de tumor primrio. a sobrevida em 60 meses nos pacientes com at 6 leses ressecadas variou de 60% a 76%, enquanto naqueles com mais de 7 ndulos ressecados, a sobrevida em 60 meses caiu para 13%. nos pacientes com doena bilateral ao diagnstico, a sobrevida global foi de 63% e naqueles com doena unilateral, essa foi de 55%. o presente estudo demonstrou que a populao analisada seguiu as caractersticas das principais casusticas na literatura : leve predominncia do sexo masculino, histologia do tumor primrio predominantemente de adenocarcinoma colorretal e aproximadamente 30% de bilateralidade da doena ao diagnstico da metstase pulmonar. () a via de acesso mais utilizada em todos os estudos a toracotomia, sendo que a abordagem dos dois pulmes no mesmo ato operatrio pouco descrita.(-) nesses casos optamos preferencialmente pela esternotomia ; porm, dependendo da localizao das leses, a bitoracotomia pode ser a melhor alternativa. a opo de videocirurgia como via de acesso ainda muito controversa na literatura para a resseco de leses metastticas, mesmo em se tratando de leso nica, devido impossibilidade da palpao cuidadosa de todo o parnquima pulmonar e limitao dos mtodos de imagem na avaliao pr - operatria. com a crescente elevao da sensibilidade dos mtodos de imagem, sobretudo naqueles casos em que ocorre a localizao perifrica das leses, podemos acreditar que os acessos minimamente invasivos podero ser mais utilizados em um futuro prximo. o tratamento cirrgico das metstases pulmonares nos principais estudos seguiu os princpios de que todas as leses devem ser totalmente ressecadas com margens cirrgicas livres de doena e que a resseco do parnquima pulmonar sempre deve ser a mais econmica possvel. dentre as tcnicas operatrias para a exrese das leses, as mais frequentemente utilizadas em vrios estudos foram a resseco em cunha e a segmentectomia, seguidas em menor nmero pela lobectomia e pneumonectomia. (,) todos esses dados foram extensamente expostos na recente meta - anlise conduzida por pfannschmidt. em 2007, na qual 20 grandes estudos sobre o tratamento cirrgico de metstases pulmonares de cncer colorretal foram comparados. () atualmente, a doena metasttica pulmonar tem a cirurgia como parte relevante de seu tratamento, e sua prtica j difundida em todo o mundo, com baixas taxas de mortalidade e aumento da taxa de sobrevida em cinco anos. () a taxa de mortalidade perioperatria variou de 0% a 2,02%, enquanto, no estudo brasileiro de younes., tais resultados apresentam boa concordncia com o nosso valor encontrado de 1,9%, demonstrando a segurana do mtodo. a sobrevida global em 60 meses em nossa casustica, de aproximadamente 56%, se mostrou semelhante s relatadas na meta - anlise de pfannschmidt. (,) entretanto, como consideramos a sobrevida a partir do tratamento do tumor primrio, observamos que vrios trabalhos tinham resultados bem menores, uma vez que calcularam a sobrevida a partir da resseco da metstase. como exemplo, temos que, no trabalho de younes., () a sobrevida em 60 meses variou de 10 - 40%, dependendo do stio primrio do tumor. a prevalncia da neoplasia colorretal como stio de origem das metstases pulmonares (47,9% em nosso estudo) nos levou a estudar em separado a sobrevida global desse grupo em comparao daqueles com neoplasia musculoesqueltica e em outros stios anatmicos. encontramos que a melhor sobrevida em 60 meses foi no grupo de pacientes com neoplasia colorretal (68%), enquanto a pior sobrevida foi no grupo de pacientes com neoplasia musculoesqueltica (26%). a grande maioria dos estudos avalia exclusivamente a doena secundria neoplasia colorretal e traz valores de sobrevida global em 60 meses variando de 24,0% a 62,7%. (,) dessa forma, julgamos necessrios mais estudos comparativos de tratamento de metstase pulmonar de neoplasia colorretal com aquele de metstase pulmonar de outros stios anatmicos que so menos frequentes a fim de poder definir com preciso o real impacto e benefcio dessa opo teraputica para grupos especficos. em nosso estudo, a sobrevida em 60 meses nos pacientes com at 6 leses ressecadas variou de 60% a 76%, enquanto naqueles com mais de 7 ndulos ressecados, a sobrevida em 60 meses caiu para 13%. a maioria dos trabalhos avalia a sobrevida estratificando a presena de uma leso ressecada versus a de mltiplas leses ressecadas, encontrando melhores resultados nos pacientes com leses nicas. (,,) entretanto, esses mesmos autores levantam os resultados controversos de vrios estudos que no encontraram diferenas significativas na sobrevida de pacientes com mltiplos ndulos, (,) podendo essa diferena de resultados residir na heterogeneidade do comportamento biolgico dos diferentes tipos histolgicos dos tumores estudados, sugerindo a necessidade de estudos mais amplos e especficos em relao a essa varivel. poucos estudos abordam a questo da lateralidade das leses ao diagnstico da metstase e a necessidade de mais de um ato operatrio para ressecar as leses. em nosso estudo, tivemos que 35 pacientes (29,4%) foram diagnosticados com doena bilateral e que 28 foram submetidos a mais de um ato operatrio (sendo que 1 paciente foi submetido a quatro procedimentos cirrgicos). na literatura, temos que a resseco de novas leses de metstases pulmonares mostra um aumento na sobrevida de pacientes submetidos a mais de um procedimento cirrgico em relao queles tratados com cirurgia nica (48% vs. 34%), () o que nos impulsiona a acreditar no benefcio e a manter a indicao do tratamento cirrgico das leses recorrentes desde que o paciente apresente condies clnicas para o ato operatrio. com relao ao tratamento de pacientes portadores de leses bilaterais, a sobrevida global que encontramos foi de 63% no grupo com doena bilateral ao diagnstico e de 55% no grupo com doena unilateral, ou seja, sem diferena estatisticamente significante, concordando com os poucos estudos que abordam esse fator e corroborando a importncia da resseco na doena bilateral. () outro fator que se destacou nas anlises de sobrevida global foi a piora na sobrevida dos pacientes que realizaram quimioterapia / radioterapia em comparao aos que no fizeram. entendemos esse dado como significado de que a necessidade de tratamento neoajuvante e adjuvante indica doena em estgios mais avanados e debilidade que envolve o tratamento multimodal sequencial. () que encontraram uma melhora na sobrevida no grupo de pacientes submetido a quimioterapia / radioterapia, assim como a ausncia de diferenas significativas entre esses dois grupos nos principais estudos(-,) levantados na meta - anlise de pfannschmidt, () o que nos leva a concordar com a opinio daqueles autores de que os diferentes protocolos para os diferentes tipos de tumor primrio podem dificultar o entendimento do real impacto na sobrevida de pacientes submetidos ao tratamento complementar. poucos so os estudos que avaliam a presena de complicaes perioperatrias e sobrevida desse especfico grupo entretanto, no houve a definio dos parmetros utilizados para a classificao desses eventos nem uma anlise da sobrevida exclusiva nesse grupo. na casustica apresentada no presente estudo, a taxa de complicaes (entre maiores e menores) foi de 22%, e a anlise de cox confirmou a piora do prognstico para esse grupo de pacientes. (como, por exemplo, escape areo por mais de cinco dias) como uma potencial morbidade, salienta - se a importncia de que mesmo esses eventos, de forma isolada ou cumulativa, podem ser preditivos de um pior prognstico para o paciente. conclumos que o tratamento cirrgico das metstases pulmonares oriundas de diferentes stios tumorais efetivo e seguro, com significativa sobrevida global, especialmente nos casos com um menor nmero de leses pulmonares. observou - se menor sobrevida nos casos de metstases a partir de sarcomas e naqueles pacientes que foram submetidos a tratamentos quimioterpicos / radioterpicos neoadjuvantes ou adjuvantes. estudos prospectivos e multicntricos com maiores casusticas e com melhor estratificao biolgica e molecular devem oferecer uma compreenso mais refinada e novos parmetros prognsticos dessas neoplasias malignas sistmicas.	objective : to describe demographic characteristics, surgical results, postoperative complications, and overall survival rates in surgically treated patients with lung metastases. methods : this was a retrospective analysis of 119 patients who underwent a total of 154 lung metastasis resections between 1997 and 2011. results : among the 119 patients, 68 (57.1%) were male and 108 (90.8%) were white. the median age was 52 years (range, 15 - 75 years). in this sample, 63 patients (52.9%) presented with comorbidities, the most common being systemic arterial hypertension (69.8%) and diabetes (19.0%). primary colorectal tumors (47.9%) and musculoskeletal tumors (21.8%) were the main sites of origin of the metastases. approximately 24% of the patients underwent more than one resection of the lesions, and 71% had adjuvant treatment prior to metastasectomy. the rate of lung metastasis recurrence was 19.3%, and the median disease - free interval was 23 months. the main surgical access used was thoracotomy (78%), and the most common approach was wedge resection with segmentectomy (51%). the rate of postoperative complications was 22%, and perioperative mortality was 1.9%. the overall survival rates at 12, 36, 60, and 120 months were 96%, 77%, 56%, and 39%, respectively. a cox analysis confirmed that complications within the first 30 postoperative days were associated with poor prognosis (hazard ratio = 1.81 ; 95% ci : 1.09 - 3.06 ; p = 0.02). conclusions : surgical treatment of lung metastases is safe and effective, with good overall survival, especially in patients with fewer metastases.
despite of an insignificant track record of quasi market models in sweden, new models of this kind have recently been introduced in health care ; commonly referred to as choice of care. this time citizens act as purchasers ; choosing the primary care centre or family physician they want to be treated by, which, in turn, generates a capitation payment to the chosen unit. policy makers believe that such systems will be self - remedial, that is, as a result of competition the strong providers survive while unprofitable ones will be eliminated. because of negative consequences of the fragmented health care delivery, policy makers at the same time also promote different forms of integrated health care arrangements. one example is local health care, which could be described as an upgraded community - oriented primary care, supported by adaptable hospital services, fitting the needs of a local population. this paper reviews if it is possible to combine this kind of integrated care system with a competition driven model of governance, or if they are incompatible. the findings indicate that some choice of care schemes could hamper the development of integration in local health care. however, geographical monopolies like local health care, enclosed in a non - competitive context, lack the stimulus of competition that possibly improves performance. thus, it could be argued that if choice of care and local health care should be combined, patients ought to choose between integrated health care arrangements and not among individual health professionals.	purposedespite of an insignificant track record of quasi market models in sweden, new models of this kind have recently been introduced in health care ; commonly referred to as choice of care. this time citizens act as purchasers ; choosing the primary care centre or family physician they want to be treated by, which, in turn, generates a capitation payment to the chosen unit. policy makers believe that such systems will be self - remedial, that is, as a result of competition the strong providers survive while unprofitable ones will be eliminated. because of negative consequences of the fragmented health care delivery, policy makers at the same time also promote different forms of integrated health care arrangements. one example is local health care, which could be described as an upgraded community - oriented primary care, supported by adaptable hospital services, fitting the needs of a local population. this paper reviews if it is possible to combine this kind of integrated care system with a competition driven model of governance, or if they are incompatible.theoryinter-organisational and interprofessional collaboration, accessibility of services, and provider continuity.methodliterature-based review.results and conclusionsthe findings indicate that some choice of care schemes could hamper the development of integration in local health care. however, geographical monopolies like local health care, enclosed in a non - competitive context, lack the stimulus of competition that possibly improves performance. thus, it could be argued that if choice of care and local health care should be combined, patients ought to choose between integrated health care arrangements and not among individual health professionals.
increasingly, it is hypothesized that schizophrenia reflects subtle connectivity dysfunction (pettersson - yeo., 2011). converging neurophysiological and neuroimaging data have documented widely distributed abnormalities in brain activity and functional connectivity (buckholtz and meyer - lindenberg, 2012 ; friston and frith, 1995 ; pettersson - yeo., 2011 ; stephan., 2006), validating the overarching disconnection hypothesis of schizophrenia (andreasen., 1998). in terms of the structural aspects, a qualitative review (kubicki., 2005) and a quantitative meta - analysis (ellison - wright and bullmore, 2009) have documented widespread reductions in white matter fractional anisotropy in chronic schizophrenia, which have also been observed in first - episode patients (friedman., 2008). an abnormal white matter ultrastructure likely underlies abnormal cooperation among brain networks (calhoun., 2009). after finding abnormal functional connectivity between the frontal and temporal regions, friston and frith (1995) proposed fronto - temporal disconnection as a key neurobiological feature of schizophrenia dysconnectivity has been introduced to refer to abnormal integration between anatomically distinct brain regions (stephan., 2006, 2009). (1998) suggested that disruption of the cortico - cerebellar thalamic cortical (cctc) circuits underlies the deterioration in neural synchrony, with improper coordination of the mental processes leading to cognitive dysmetria. abnormal neural synchrony (ford., 2007) is hypothesized to be one of the main causes of cognitive dysfunction in schizophrenia. a recent review (uhlhaas and singer, 2010) concluded that altered neural oscillations and synchrony are crucial elements in the pathophysiology of schizophrenia. this conclusion was based on aberrant connectivity and synchrony findings in patients with schizophrenia (friston and frith, 1995 ; garrity., 2008 ; kim., 2008), as well as differences in effective connectivity in schizophrenia patients compared to controls (demirci., 2008 ; the dysconnectivity hypothesis has been extended to explain specific symptoms in schizophrenia, particularly auditory hallucinations (ah) (rish., 2013) and visual hallucinations and ah in findings by amad. neuroimaging studies have found abnormal activation in patients with ah, particularly in cortical regions of language processing (allen., 2008). several studies have shown abnormal structural and functional connectivity in patients with ah (benetti. most studies have found reduced functional connectivity between the temporal, limbic and frontal areas (hoffman., 2011 ; vercammen., 2010 few studies (e.g., mechelli., 2007) have analyzed synchrony and effective connectivity in a group of patients with schizophrenia and ah. emotional processing disturbances have been related to the origination of ah (sanjun., 2006 ; aleman and lari, 2008). our group has focused on understanding emotional processing in patients with ah. in a previous study, we obtained evidence of enhanced activation of the limbic and frontal brain areas in a small group of patients with persistent ah engaged in passively listening to emotional words (sanjun., 2007). these results implicated circuits subserving the processing of emotional stimuli as a neural substrate of ah. in a subsequent work, we studied functional connectivity in response to an emotional auditory paradigm in patients with ah by conducting independent component analyses (ica) (escart., 2010). using this approach, the activated areas could be obtained by selecting the ica components related to the emotional auditory paradigm. functional connectivity is then characterized by detecting statistical dependencies among the time courses of the areas activated in response to the auditory emotional stimuli (escart., 2010). in addition to the temporal, frontal and parietal networks detected in all subjects, in the schizophrenia patients with ah, we observed a specific pattern of functional connectivity involving activation of the limbic structures (predominantly the amygdala and parahippocampal gyrus). we used an emotional auditory task in this work to analyze the synchrony patterns and granger - causal relationships to study effective connectivity and to determine which brain regions directly influence other brain regions. we predicted the following results : compared to the controls and schizophrenia patients without ah, the schizophrenia patients with ah would 1) have abnormal synchrony between the detected networks and concrete abnormalities in the frontal cortex compared with the other brain regions and 2) have different granger - causal interactions between the frontal, parietal, temporal, limbic and cerebellar networks. a total of 41 male patients with schizophrenia (27 with chronic ah and 14 without ah) were recruited. the patients with chronic ah fulfilled the following selection criteria for persistent hallucinations (sanjun., 2007) : (a) they heard voices that were resistant to treatment for at least 1 year ; (b) the voices were present at least once a day during the last year ; and (c) at least two antipsychotic drugs had been tried in the last year at doses equivalent to at least 600 mg / day of chlorpromazine. patients with a history of traumatic brain injury, epilepsy or other neurological or psychiatric history were excluded. the patients were clinically assessed with the positive and negative syndrome scale (panss) (kay., 1987), the brief psychiatric rating scale (bprs) (ventura., 1993) and the psychotic symptom rating scale (psyrats) for ah (haddock., 1999) in the 24 h prior to scanning. the participants were male, right handed (as assessed with the edinburgh questionnaire, oldfield, 1971) and caucasian, and they provided written informed consent to participate in the experiment. the healthy controls and patients did not differ significantly in terms of sex, laterality, or ethnicity. the groups differed significantly in educational level, with a greater proportion of individuals with university and high school diplomas (or equivalence) in the control group, as expected. a 1.5 tesla scanner (philips medical systems) was used to acquire bold contrasts during a stimulation paradigm (escart. an emotional auditory paradigm was designed to evoke emotions related to the patients ' hallucinatory experiences (supplementary material 1). the activation blocks in one session consisted of 13 spanish words with high emotional content. the other session had activation blocks containing 13 words with neutral or low emotional content. four blocks of stimuli (20 s each) were interleaved with four blocks of rest of 20 s each. the subjects were informed before the test regarding the two types of words they were going to hear and were asked to focus their attention on these words. pre - processing of the functional data was performed using spm (http://www.fil.ion.ucl.ac.uk/spm) (supplementary material 2). the ica analysis was performed using the group ica approach fmri toolbox (gift, http://www.icatb.sourceforge.net). components of interest (coi) were selected (in terms of the individual beta values related to the emotional task), and the regressors in each component were entered into a one - sample t - test with a threshold of p < 0.01 (escart., 2010). a correlation test was performed to evaluate the relationship between age and the components of interest loads. we used the intra - group ica time - course (tc) correlation coefficient matrix obtained for all pair - wise combinations of ica - tc to examine functional connectivity across the three groups (jafri., 2008). to observe the relationships between every coi pair, we calculated pearson 's correlation coefficients (friston, 1994) (supplementary material 3). the data tool granger causal connectivity analysis (gcca) was used to analyze causality (seth, 2010). a key challenge when analyzing such data is to study the causal connectivity using a driven - data methodology to study multivariate time series. this kind of methodology combines graph - theoretic and network - theoretic techniques that allow their quantitative characterization (seth, 2005). within a given causal network, we defined the causal flow of a node, i, as the difference (weighted or not) between its inflows and outflows. a node with positive causal flow exerts strong causal influence on a dynamic system as a whole. in contrast, a node with negative causal flow is called a causal sink (seth., 2011). the causal density of a dynamic system provides an overall measure of causal interactivity (seth, 2005). this causal density measure is defined as a causal graph for all pairs of elements in the system. those interactions that are not significant are assigned zero values, as calculated by the following expression:(1)cd(x)=1n(n1)ijfxixj\|x\|ij\| where x[ij ] is a x subsystem omitting variables xi and xj. this value could be calculated between 0 and 1 and is based on causal density, in which all significant interactions are assigned the value 1. causal density measures dynamic complexity because it reflects the complexity of integration and the coexistence of dynamic segregation (sporns, 2007). however, the elements are dynamically different ; therefore, diverse elements contribute in a different manner. i is a value related to the causal interaction of that node i normalized by the number of nodes. a system with n - nodes has n elements with n - cdn values, is an unweighted version and is calculated by assigning all significant interactions the value 1. a node with a high value for cdn as previously reported (escart., 2010), the following coi were identified in the control group : temporal (ce19), fronto - temporo - parietal (ce18), subcortical the following coi were identified in the patient group with ah : temporal (he17), fronto - parietal (he14), fronto - temporal (he11), limbic (parahippocampal amygdalar) (he06), and occipito - cerebellar (he02). the following coi were identified in the patients with schizophrenia but without chronic ah : temporal (nhe19), fronto - temporal (nhe18), subcortical temporo - parietal cerebellar (nhe13) and fronto - parietal (nhe12)., higher correlations were found for the temporal component (ce19), particularly in the relationships to the subcortical fronto - temporal (ce09) and occipito - cerebellar (ce04) components. the minimal correlation value was found in the relationship between the fronto - temporo - parietal (ce18) and subcortical fronto - temporal (ce09) components. 1a shows that the temporal (ce19) and subcortical fronto - temporal (ce09) components were synchronized, whereas the fronto - temporo - parietal (ce18) and occipito - cerebellar (ce04) components were delayed, compared with components (ce19, ce09). 1a (right) depicts the correlation function calculations for the control group in terms of the phaser graphs. the lag at which this value is obtained is represented by the means of the angle between the vector and the horizontal axis. as shown in table 3 and fig. 1b, we found high correlations among the temporal (he17), fronto - parietal (he14) and fronto - temporal (he11) components. additionally, the limbic (he06) and occipito - cerebellar (he02) components were significantly correlated with one another ; however, they were unrelated to the temporal, fronto - parietal, and fronto - temporal components (the left side of fig. the phasers corresponding to the limbic (he06) and occipito - cerebellar (he02) components (the right side of fig. 1c, we found high significant correlations between the temporal (nhe19), fronto - parietal (nhe12), subcortico - fronto - temporal cerebellar (nh13) and fronto - temporal (nhe18) components. 1c depicts the correlation function calculations for the patients without ah in terms of the phaser graphs. 1c shows that the temporal (nhe19) and fronto - temporal (nhe18) components were synchronized, whereas the frontal parietal (nhe12) and subcortico - temporo - parietal cerebellar (nhe13) components were delayed compared with the first two components. based on the granger - causality analysis, the temporal (ce19) and fronto - temporo - parietal (ce18) components were causal sources, whereas the subcortical fronto - temporal (ce09) and occipito - cerebellar (ce04) components were causal sinks (fig. the analysis of the causal density units showed that all nodes were highly integrated in the system, with the temporal (ce19) component being the main causal hub. the temporal (ce19) component exerted the highest granger - causality influence on the remaining system components. 2b, the occipito - cerebellar (he02) component was a strong causal source, whereas the fronto - parietal (he14) and limbic (he06) components were causal sinks. the graph for the patients with ah shows a large number of interactions among the components. we observed that the temporal component (nhe19) was a causal source, whereas the fronto - parietal (nhe12) and subcortico - fronto - temporal cerebellar (nhe13) components were causal sinks (fig. 2c). the temporal (nhe19) component exerted the highest granger - causality influence on the remaining system components. in a homogeneous sample of patients with schizophrenia and persistent ah, we found (1) abnormal synchrony compared to a healthy control group and to patients with schizophrenia without chronic ah, specifically between the cerebellum - limbic and frontal temporal parietal networks, as well as (2) a different pattern of effective connectivity between these functional networks while processing emotional auditory stimuli. temporal limbic cerebellar areas) in the patients compared to the controls aligns with reports of reduced frontal connectivity (pettersson - yeo., 2011) and disconnections between the frontal and temporal lobes (calhoun., 2004 ; garrity., 2007 ; kim., 2008). we used the task of passively listening to emotional auditory stimuli, which elicited a different pattern of functional response between the controls and patients because we only observed activation in a network comprising subcortical limbic areas (amygdala and parahippocampal gyrus) in the patients with ah ; these results have been previously reported and discussed in depth (escart., 2010). this analysis showed that the subcortical limbic network and an occipito - cerebellum network were more synchronized in the patients with ah (particularly in the fusiform gyrus, culmen and declive of the vermis) and desynchronized from the frontal parietal temporal areas. the controls and patients without ah showed more synchronization between the networks (frontal parietal temporal limbic cerebellar areas) than the patients with ah. our results align with the most common finding in the literature, abnormalities in the frontal lobe (fornito., 2011 ; salvador., 2010 ; zalesky., 2011 ; zalesky., 2010) and prefrontal regions connections with other brain regions, including the cerebellum and the parietal and occipital cortices. additionally, altered regional connectivity of the parietal, temporal and occipital cortex, as well as the subcortical nuclei, has been observed (lynall., 2010). it is hypothesized that language dysfunction located in the frontal and temporal regions and specific dysconnectivity between the two regions play critical roles in the core pathophysiology of schizophrenia studies, particularly in studies of auditory verbal hallucinations (hubl., 2004 ; seok., our second finding was that causal interactions between the functional brain networks differed among the three groups. in the control group and the patient group without ah, we found that the main causal source was the temporal lobe network component (ce19), which aligns with previous studies of emotional auditory processing (phillips., 2003). in the control group, the temporal (ce19) and fronto - temporo - parietal (ce18) components were causal sources ; however, in the group of patients without ah, only the temporal component (nhe19) was a casual source. in the group of patients with ah, the main causal source was the occipito - cerebellar network component (he02), despite the processing of auditory emotional stimuli by the patients. the study of synchrony could inform our understanding of the functional organization of the brain, particularly in task - evoked processes (bartels and zeki, 2005 ; mckiernan. our findings support and extend the findings of numerous studies that have identified similar regions (cerebellar and limbic) associated with emotional deficits in patients with schizophrenia (aleman and kahn, 2005 ; andreasen and pierson, 2008)., the cerebellum could be considered to be crucial for synchrony (picard., 2008 ; schutter and van honk, 2005). additionally, the cerebellum is involved in higher cognitive functions (schmahmann, 2001 ; andreasen and pierson, 2008). the cerebellar circuits process learning (particularly error - related), timing and prediction in relation to motor and cognitive information (d'angelo and casali, 2012). the timing hypothesis postulates that the cerebellum is critical for representing temporal relationships among task - relevant events, which is closely related to the concept of synchrony. in this sense, the cerebellum function is analogous to a supramodal internal timing unit (such as a metronome) (ivry., 2002). hypothetically, when the cerebellar timing function is disrupted, the information processing stream becomes desynchronized, providing a nurturing environment for a diverse range of psychopathological conditions (schutter and van honk, 2005). regarding schizophrenia, the cerebellum is connected to many regions of the cerebral cortex by the cortico - cerebellar thalamic cortical circuits and might play a crucial role in this distributed circuit to coordinate or modulate aspects of cortical activity (picard., 2008). according to the cognitive dysmetria or models of schizophrenia, aberrant cerebellar modulation of information to the cerebral cortex is involved in the pathophysiology of the disorder (andreasen., 1998 ; schmahmann, 1998a). (2005) study, which preliminarily reported that, compared with controls, patients with schizophrenia demonstrated deficits in cerebellar inhibition. their data are corroborated by our results demonstrating between - group differences in the causal flow of information between the cerebellar occipital component and other regions, with an afferent direction of flow for the controls (figs. 2a and 2c) and an efferent direction for the patients with ah (fig. there is evidence that neuroanatomical damage to the cctc could be the primary pathophysiological alteration in schizophrenia (konarski., 2005). it is well known that cerebellar abnormalities exist in schizophrenia patients (andreasen and pierson, 2008). our findings suggest that the functional dysconnectivity of the cerebellum with the cerebrum predominantly involves the anterior and vermal areas of the cerebellum, the main areas included in the occipito - cerebellar component in the group of patients with ah. the vermis has previously been labeled as the limbic cerebellum because the anterior vermis is the principal cerebellar target of limbic projections (schmahmann, 2000). behavioral studies have supported a relationship between the cerebellar midline structures and the modulation of emotion (heath and harper, 1974 ; stoodley and schmahmann, 2010), and lesions of the vermis have been shown to produce affective symptoms ranging from emotional blunting and depression to disinhibition and psychotic features (schmahmann and sherman, 1998b). (2010), who found less activation of the left cerebellum, right insula and left parahippocampal gyrus in patients with ah (2014), which showed decreased connectivity in several brain areas in patients with ah, including between the right cerebellum and left thalamus. in this study, we relied on ica, a driven - data method that could separate independent spatial temporal patterns of neural activity from the fmri data in a manner that has been helpful in studying intrinsic brain networks (damoiseaux., 2006). granger - causality was used to explore effective connectivity in fmri data to quantify the strength of interactions between activated brain areas (goebel., 2003 ; demirci., 2009 ; londei., 2007) the ica and granger - causality techniques allowed us to analyze the functional and effective connectivities, respectively. ica allowed us to select the coi that were candidates for the gcca analysis of their associated time - courses. the main strength of this methodological framework was the combination of these independent techniques, which allowed us to observe whether different methods would lead to consistent results and more reliable conclusions. first, all patients were taking antipsychotics at the time of scanning, although we did not find any significant correlations between the chlorpromazine equivalents and our imaging measures. second, our small sample size limited our power to detect relationships with symptom severity or duration of illness, our findings of impaired synchrony should be confirmed in larger samples, preferably with unmedicated first - episode schizophrenia patients. third, the sample of patients without ah (n = 14) is relatively small compared with the control group (n = 31) and patients with ah (n = 27) groups. including a subgroup of schizophrenia patients treated with the identical medications and differing only in the absence of persistent ah strengthens our confidence in our findings. fourth, the application of the granger - causality method to the fmri data is somewhat controversial (friston, 2009 ; roebroeck., 2009). specifically, granger - causality analysis ignores the influence of other areas when assessing whether coupling between reference regions a and b is driven by region c (gao. accordingly, we employed gcca (geweke, 1984), which is based on a direct expansion of the autoregressive model to a multivariate general case, including all measured variables. patients with schizophrenia and ah exhibit abnormal patterns of neural synchrony, as well as different patterns of granger - causal interaction between functional networks compared with controls and patients with schizophrenia without ah. the results indicate an anomalous process of neural connectivity in the cortico - cerebellar thalamic cortical circuits in patients with ah. a central role for the cerebellum in the pathological processing of emotional stimuli by patients with schizophrenic and persistent ah is suggested, perhaps reflecting deficiencies in predicting the emotional effect of a given stimuli.	auditory hallucinations (ah) are the most frequent positive symptoms in patients with schizophrenia. hallucinations have been related to emotional processing disturbances, altered functional connectivity and effective connectivity deficits. previously, we observed that, compared to healthy controls, the limbic network responses of patients with auditory hallucinations differed when the subjects were listening to emotionally charged words. we aimed to compare the synchrony patterns and effective connectivity of task - related networks between schizophrenia patients with and without ah and healthy controls.schizophrenia patients with ah (n = 27) and without ah (n = 14) were compared with healthy participants (n = 31). we examined functional connectivity by analyzing correlations and cross - correlations among previously detected independent component analysis time courses. granger causality was used to infer the information flow direction in the brain regions.the results demonstrate that the patterns of cortico - cortical functional synchrony differentiated the patients with ah from the patients without ah and from the healthy participants. additionally, granger - causal relationships between the networks clearly differentiated the groups. in the patients with ah, the principal causal source was an occipital cerebellar component, versus a temporal component in the patients without ah and the healthy controls.these data indicate that an anomalous process of neural connectivity exists when patients with ah process emotional auditory stimuli. additionally, a central role is suggested for the cerebellum in processing emotional stimuli in patients with persistent ah.
esophageal cancer is one of the least common and most deadly gastrointestinal tract neoplasms. in 2010 in poland, the standardized incidence ratio for esophageal cancer was 3.3/100,000 for men and 0.6/100,000 for women. patient survival is short, and the incidence / mortality ratio for both sexes was 0.8 in 2010. for many years, the primary method of treating patients with advanced esophageal cancer (ec) has been surgical treatment, which is associated with significant injury. the surgery is burdened with the highest perioperative mortality rate among all gastrointestinal tract procedures, reaching 18 - 20% according to some reports [35 ]. technological development as well as improved preoperative assessment, surgical technique, and postoperative care play a significant role in the improvement of surgical treatment outcomes. the premise of minimally invasive techniques in esophageal surgery is to maintain the therapy effectiveness and quality of traditional operations while reducing perioperative injury. the techniques are based on the principle of delicate dissection in order to minimize the damage. moreover, minimally invasive techniques are widely accepted by patients and medical personnel. reducing the negative psychological impact of open surgery leads to the improvement of patient satisfaction from the employed treatment. the first reports of thoracoscopic esophagus removal were published over 20 years ago [7, 8 ]. the premises of thoraco - laparoscopic minimally invasive esophagectomy (mie) remain identical as those of open surgery (oncological radicality r0, 2- or 3-field lymphadenectomy). however, certain reservations remain concerning the oncological value of mie as well as the risks and costs related to these technically demanding and time - consuming surgical procedures. the key factor of mie is the proper qualification of patients, which should be based on the precise evaluation of the stage of the neoplastic disease. the patients undergo diagnostic examinations : endoscopic ultrasound and computed tomography of the chest and abdominal cavity. some centers also employ full - body positron emission tomography (pet). in patients in whom, laparoscopy is particularly useful in the case of lower esophageal adenocarcinoma ; it is also more sensitive in the diagnosis of pathological lymph nodes as well as omental, peritoneal, and hepatic metastases. it is a safe procedure with a low rate of complications ; concurrently, in the case of diffuse neoplastic disease, it allows the avoidance of redundant surgical procedures and expedites the start of palliative treatment. in the case of proximal tumors, the use of thoracoscopy in combination with pleural or mediastinal lymph node biopsy also improves preoperative evaluation by approximately 20% with regard to confirming lymph node or distal metastases. most frequently, the method can be used in patients with persistent dysphagia resulting from digestive stenosis, end - stage achalasia, or extreme insufficiency of the esophageal passage in other functional diseases of the esophagus [10, 11 ]. indications for the use of mie techniques in cancer patients remain more controversial. in cases in which extensive lymphadenectomy is not required, mie appears to be an ideal technique for treating changes such as severe dysplasia [12, 13 ] ; however, in such cases, advanced endoscopic techniques remain a popular alternative. minimally invasive esophagectomy is more acceptable for invasive cancer ; it is comparable to traditional resections in terms of outcome and causes less perioperative damage. the contraindications for mie include massive pleural adhesions, previous lung surgery, extensive tumors, and local infiltration, especially of the respiratory system. extensive adhesions after abdominal surgery may constitute a contraindication for laparoscopy. due to the necessity of prolonged one lung ventilation, the respiratory and circulatory systems of patients qualified for mie should exhibit proper efficiency. at present, there is still no clear consensus concerning the preferred operative technique in esophageal surgery. transhiatal esophagectomy (the) and transthoracic esophagectomy (tte) are complex procedures which are usually employed in the treatment of patients suffering from esophageal cancer. as with open procedures, in the case of mie, there is no agreement as to which specific operative method is superior. the most commonly used techniques are presented in table i. the most important development in mie was achieved by luketich., who employed a thoracoscopic technique to dissect the esophagus with the patient lying on the left side and performed laparoscopic mobilization and reshaping of the stomach with the patient lying supine followed by typical neck anastomosis. notwithstanding, most authors suggest selecting the mie technique individually for each patient in order to avoid intraoperative problems and complications. in the case of tumors located in 1/3 of the upper thoracic esophagus, it is justifiable to employ the thoracoscopic approach, while the use of the laparoscopic transhiatal technique is warranted for the distal esophagus. laparoscopic surgical tools are introduced through 5 mm ports : one located in the right subcostal region and two in the left subcostal region. subsequently, the stomach is pulled upwards, and the left gastric vessels are cut with a vascular stapler. a linear stapler is used to construct a gastric tube. feeding jejunostomy is introduced through one of the left abdominal trocar holes. after the abdominal stage of the surgery is completed, the patient is placed on the left side, and right lung ventilation is turned off. during dissection, after its removal, the next stage of the surgery consists in creating an anastomosis. this can be achieved with both staplers and manual suturing techniques. mechanical anastomosis is often performed by means of a circular stapler introduced through the patient 's mouth. the risk of a leak in an anastomosis created in this manner does not exceed 10% and is comparable with other stapler techniques. the gastroesophageal anastomosis is tightened with a pedicled greater omental flap. there are no mie experiences involving the reconstruction of gastrointestinal tract continuity using other substitutes (the colon or small intestine). according to hoppo., laparoscopic surgery with esophageal invagination performed through a neck incision (stripping) is the least invasive technique for esophagectomy ; its more commonly used variant is laparoscopic inversion esophagectomy (lie). this type of surgery may always be considered in benign diseases of the esophagus, severe dysplasia, t1n0 tumors, and in view of contraindications for thoracotomy. for locally advanced cancer of the middle and upper segments of the thoracic esophagus, the author recommends 3-field mie in the following order : abdominal stage, thoracic stage, neck anastomosis. for tumors in the lower segment of the esophagus, minimally invasive operations must oncological criteria for esophagus removal, which are also used in the case of open surgery. above all, the surgery must adhere to the principles of oncological radicality with the preservation of distal, proximal, and radial (r0) margins, two / three - field lymph node dissection, and oncological asepsis of the laparo / thoracoscopic ports. the specific type of surgery should be selected individually depending on the type of cancer, tnm staging, and the availability of endoscopic methods. patients undergoing mie operations should be monitored and followed up in the same manner as those undergoing traditional surgery [20, 21 ]. employing laparoscopic or thoracoscopic access in esophagectomy has its disadvantages related to, e.g., the limitations of the used instruments, narrow operative space in the mediastinum, and 2d imaging. the introduction of robotic techniques (3d imaging, articulated surgical tools) has created an opportunity for a significant improvement of mie operations. robotically assisted techniques may be employed during the thoracic dissection of the esophagus, gastric mobilization, and the performance of thoracic anastomosis. it may also be used in combination with laparoscopy, manually assisted laparoscopy, or thoracoscopic access. the robotic technique employs a similar set of laparoscopic ports, using 5 mm trocars instead of 8 mm ones. in the available literature, the analyzed groups of patients undergoing mie are small, and the published reports are mainly retrospective comparative studies. the data gathered by the authors revealed longer operating times associated with mie procedures in comparison with hmie (hybrid minimally invasive esophagectomy ; thoracoscopy and laparotomy) and traditional surgery, lower numbers of excised lymph nodes in mie / hmie operations in comparison with the open technique, shorter hospitalization time for mie in comparison with hmie and open surgery, as well as lower rates of pulmonary complications and anastomotic leaks in the mie group. the analyzed material included 1011 patients with esophageal or gastric cardia cancer undergoing video - assisted surgery in the years 1996 - 2011. perioperative mortality was 2.8%, while the rate of severe complications did not exceed 6%. the location (distal esophagus / cardia) and histological type of neoplasms (primarily adenocarcinoma) caused the medical team to change the operative strategy most procedures conducted after 2006 were mie with thoracic anastomoses. as a result, a decrease in the frequency of recurrent laryngeal nerve palsy was noted from 8% to 1%. in the analysis of 481 procedures (48%) with cervical anastomosis and 530 procedures (52%) with thoracic anastomosis, no differences were found in perioperative mortality and the frequency of other perioperative complications. oncological radicality was achieved in both operative techniques in 98% of patients ; the mean number of excised lymph nodes was 21. these figures are comparable to the best results achieved in experienced centers performing open esophageal resections. these data prove that mie is a safe method, improving the postoperative course and resulting in shorter hospital stay. conducted a meta - analysis comparing two groups of operations : 1 operation via thoracoscopic - laparoscopic approach (mie) vs. right - sided thoracotomy with laparotomy, 2 thoracoscopic approach with laparotomy (hmie) vs. right - sided thoracotomy with laparotomy. no significant differences in terms of serious approach - dependent postoperative or pulmonary complications were found in group 1. in group 2, a tendency toward lower postoperative mortality among patients undergoing mie / hmie was found in both groups. decker. presented the results of an analysis comparing various approaches for esophagectomy. the authors did not report any differences between the analyzed methods. according to butler., the mie thoracic approach provides a significantly better visualization of the esophagus when compared to the transhiatal approach, making it the preferred option. dantoc., on the basis of the results of 17 publications, estimated that the mean number of lymph nodes excised in mie operations was higher than in the case of traditional operations ; however, this did not have any influence on the patients survival. another meta - analysis, performed by nagpal., concerned the results of mie resections and traditional esophagectomy. the authors did not find any differences in perioperative mortality rates in a group of 672 patients after mie and 612 patients after traditional resection. the patients after mie were hospitalized for shorter periods of time, exhibited lower blood loss, and suffered from fewer pulmonary and postoperative complications in total. the results of the first multi - center prospective randomized study comparing the outcomes of mie with open esophagectomy due to esophageal cancer were published in 2012. the research was conducted in five european centers performing at least 30 esophageal resections a year. a group of 56 patients underwent open surgery, while a group of 59 patients underwent mie. respiratory complications were found in 16 patients (29%) operated on with the traditional method and in 5 patients (9%) after mie (p = 0.005). one patient undergoing traditional surgery died as a result of a fistula in the anastomosis. the acquired results prove the significant benefits of mie in the treatment of patients with resectable esophageal cancer. this study is the only methodologically correct, multi - center clinical study of mie. it is limited by the lack of long - term outcomes and the fact that its results need to be confirmed by other multi - center, randomized studies. published the results of a retrospective analysis of the first 50 mie procedures performed using the robotically assisted ivory - lewis method. the obtained results suggest that robotically assisted esophagectomy is at least as oncologically effective as open surgery. the authors emphasize that such procedures should be performed in specialized centers with a lot of experience with both open esophagectomy and mie. despite its tremendous benefits associated with dissecting precision, this modern technique the potential benefits and controversies related to the implementation of mie operations in clinical practice are presented in table ii. minimally invasive esophagectomy techniques performed in reference centers conducting large numbers of such procedures constitute an important alternative in the surgical treatment of esophageal cancer patients. mie is associated with lower blood loss, less postoperative pain, and shorter hospital and icu stay. there are no detailed data available on the survival time of patients after mie, which results from the relatively short period of observation. no significant differences in the survival of patients after traditional surgery and mie have been found to date. the significant limitations of mie include longer operating time, high cost, and low availability of medical equipment (tools, staplers, robots). the risk of trocar site metastasis should also be taken into consideration. moreover, mie techniques are not subject to standardization. the learning curve is long and the number of complications in a given center may initially be higher. it is estimated that a surgeon has to perform at least 30 or even 50 mie operations to achieve sufficient surgical expertise. the establishment of a single standpoint regarding the selection of an optimal approach in esophagectomy is complicated by the diversity of the available mie techniques. such research should result in the recommendation of a single procedure with the lowest rate of recurrence and complications and the best postoperative quality of life, which would provide an optimal alternative for the surgical treatment of patients with esophageal cancer.	open esophagectomy (oe) requires extensive surgery and is associated with significant morbidity and mortality. furthermore, the long - term results of esophageal cancer surgery are not satisfactory ; hence, the best surgical approach is constantly under debate. during the last twenty years, minimally invasive esophagectomy (mie) employing laparoscopy and/or thoracoscopy has been introduced in a growing number of centers worldwide. to date, several studies have demonstrated that mie has better outcomes than oe, as it results in shorter hospital stay and decreased overall morbidity. however, the length of operating time in mie is increased in comparison to oe. the survival benefit has been demonstrated to be similar in oe and mie. highly advanced laparo - thoracoscopic skills are required to perform mie ; along with the relatively long learning curve, this makes mie feasible only in high - volume, experienced university surgical centers. there is a need for further large - scale comparative studies to prove the superiority of mie over open surgery.
obesity - related comorbidities seem to be more closely related to body fat distribution (e.g., upper versus lower, visceral versus subcutaneous, and truncal versus peripheral) rather than the total amount per se. abdominal obesity is a relevant predictor of a higher metabolic risk, assuming that insulin resistance (ir) is the common link between visceral adiposity and dyslipidemia [24 ], type 2 diabetes mellitus (dm) [5, 6 ], liver fat storage, hypertension, and other cardiovascular diseases (cvd) [810 ]. two major pathophysiological hypotheses have been proposed to explain the dysmetabolic milieu observed in abdominal obese individuals. it has been proposed that neuroendocrine perturbations, mediated by hypothalamic - pituitary - adrenal (hpa) axis stimulation, are responsible for ir and abdominal obesity [11, 12 ]. alterations in cortisol secretion, inhibition of steroid and growth hormones production, and stimulation of sympathetic nervous centers are some of the dysfunctions which may precipitate metabolic disturbances. conversely, according to the portal hypothesis, the increased lipolytic activity in visceral adipocytes leads to an augmented release of free fatty acids (ffa) into portal circulation, promoting liver fat storage that is accompanied by hepatic metabolism disturbances and ir [6, 13, 14 ]. in this context, abdominal obesity has been associated with ectopic fat storage, defined as fat accumulation outside classical depots such as heart, skeletal muscle, pancreas, and liver. liver fat is associated with obesity, increased concentrations of plasma ffa, as well as with the ir degree, both in obese and type 2 dm patients [14, 16, 17 ]. furthermore, a lower liver - to - spleen ratio (lsr), a reliable index of liver fat, has been independently associated with visceral adiposity [7, 19, 20 ], hepatic ir [6, 17, 19, 21 ], dyslipidemia, and several other metabolic syndrome features [6, 7, 19, 22 ]. additionally, hepatic steatosis has also been associated with ir and major cvd risk factors due to a combination of abnormalities including increased liver ffa influx and synthesis, decreased ffa oxidation, increased very low - density lipoprotein (vldl) particles secretion, and a low - grade chronic proinflammatory state [21, 23 ]. although evidence has highlighted the independent contributions of both visceral adiposity and liver fat to an increased metabolic risk in obese and type 2 dm patients, it is not totally clear if liver fat is additionally associated with other specific inflammatory and atherothrombotic markers [6, 7, 19, 22 ]. on the other hand, despite the recognized abdominal obesity relevance to ectopic fat storage, little is known about the relationships of other fat compartments, such as thigh at, with liver fat, and consequently with hepatic steatosis. in fact, only one study developed in type 2 dm patients has reported that thigh subfascial at was associated with both liver fat and ir features. therefore, based on previously defined criteria, the current study examined the independent associations of abdominal and thigh at compartments with hepatic fat in overweight and obese premenopausal caucasian women. additionally, this study investigated the associations of liver fat with metabolic, proinflammatory, and atherothrombotic risk factors. participants in this investigation were 140 premenopausal overweight and obese caucasian women, recruited from the lisbon (portugal) area by public advertisements. study inclusion criteria were the following : subjects could not be pregnant or trying to become pregnant, > 24 years, > 24.9 kg / m body mass index (bmi), were not under any medication that could affect weight, body composition or liver metabolism, had no clinical or laboratory evidence of liver or spleen disease, and had no history of cancer in the last five years. exclusion criteria were ongoing hormonal medication, history of cvd, stroke, hypertension, type 2 dm, cushing syndrome, hormonal dysfunction, resting and exercise abnormal electrocardiograms, as well as the presence of drinking habits. subjects that were undertaking oral medication to treat hypertriglyceridemia, hyperglycemia, or hypercholesterolemia were also excluded. all subjects were informed about the purpose, nature and study design before giving their full written consent to participate. the study protocol was performed according to the principles of the helsinki declaration and was approved by the human subjects institutional review board of the faculty of human movement, technical university of lisbon. height was measured to the nearest 0.1 cm with a stadiometer (seca, hamburg, germany). body weight was measured to the nearest 0.01 kg on a previous calibrated scale after removing shoes and heavy clothing. abdominal sagittal diameter (sd), waist circumference (wc), and hip circumference (hc) measurements were performed according to lohman both wc and hc measurements were taken with a stiff fibreglass tape to the closest 0.1 cm. all anthropometric variables were measured by trained technicians and repeated 3 times, with the mean value being used. bmi was calculated as weight divided by height squared (kilograms per square meter), and waist - to - hip ratio (whr) was defined as the wc divided by hc. trunk fat mass (tfm), total body fat mass (tbfm), and total body lean mass (tblm) were measured by a pencil beam mode dxa (qdr-1500 hologic, waltham, mass, usa). all measurements were made with volunteers in the supine position with their arms separated from their trunk. the same technician performed the all the scans and completed the analysis according to the operator 's manual. the intraobserver coefficient of variation (cv) for tbfm and tblm was 2.0% and 1.7%, respectively. a 0.5% technical error for % tbfm was obtained as estimated in 2 repeated measures performed on 10 subjects. with the subjects supine and arms extended above their head, a single cross - sectional ct (somaton plus ; siemens, sorheim, germany) l4-l5 intervertebral space image was acquired to measure abdominal at compartments, as described elsewhere. all images were obtained using 120 kvp, 480 ma, and 512 512 matrix with a 48-cm field of view. total abdominal adipose tissue (taat), abdominal subcutaneous adipose tissue (ab sat), superficial and deep ab sat, and visceral at (vat) areas were measured. the boundary between vat and ab sat was defined using the abdominal and oblique muscles in continuity with the deep fascia of the paraspinal muscles and the anterior aspect of the vertebral body. the subcutaneous fascia was used to differentiate ab sat into its superficial and deep compartment. using the same scan parameters, contiguous 7-mm - thick cross - sectional images of both legs were obtained between the inferior ischial tuberosity and the superior border of the patella. total thigh adipose tissue (ttat), total thigh subcutaneous at (ttsat), thigh subfascial at (ttsfat), and muscle tissue areas and attenuations were measured. the tissues volumes (cubic centimetres) identified in each image were calculated by multiplying the image thickness (7 mm) by the tissue area (square centimetres). thigh at volume (litters) was then converted to mass units (kilograms) multiplying the volume by the assumed constant of fat density (0.92 total thigh muscle mass was also calculated multiplying its volume by the constant density assumed for at - free skeletal muscle (1.04 the thigh scans performed, it was selected a single slice located at the mid - point distance between both anthropometric markers previously described to image mid - thigh at and muscle tissue distribution. a 7-mm - thick cross - sectional image at t11-t12 intervertebral space was acquired to measure both liver and spleen ct attenuations, which were determined by calculating the mean hounsfield units (hus) of three regions of interest (roi) (liver roi had 120 mm, located 2 in right lobe and 1 in left lobe ; spleen roi had 75 mm). as previously described, all roi were consistently selected in peripheral parenchyma areas, away from artefacts, major blood vessels and other areas of inhomogeneity. the ratio of mean liver to spleen attenuation values, defined as liver - to - spleen ratio (lsr), has been defined as reliable index of liver fat. fatty liver was present when lsr 0.05). on the contrary, both weight and bmi were inversely associated with lsr, even when adjusting for age (= 0.235, p 0.05). our primary findings were that, in a sample of overweight and obese premenopausal women, a higher thigh sfat area was associated with a higher lsr, representing a lower liver fat storage, independently of age and bmi. additionally, we found that for a given wc, increased thigh sfat areas were also significantly related with higher lsr values. to our knowledge, these associations between thigh sfat and both lsr and liver attenuation are novel observations that may suggest an indirect preventive role of this thigh at depot against ectopic liver fat storage, and therefore, against hepatic steatosis, in overweight or obese premenopausal women without type 2 dm. it has been suggested that femoral - gluteal at compartments may function as a sink for circulating ffa. when compared with visceral adipocytes, these thigh adipocytes are less sensitive to stimulated lipolysis and reveal a relatively higher lipoprotein lipase activity, important in ffa uptake from the circulation. these metabolic characteristics may prevent liver lipotoxicity and counteract the inevitable physiologic cascade observed in abdominal obese subjects, responsible for ir and other secondary metabolic disturbances, such as a multiple proinflammatory cytokine response. in this context, several studies have been reporting that peripheral fat mass (pfm), measured by dxa (unable to differentiate the different thigh adipose tissue compartments), is an independent predictor of a lower cardiovascular risk [32, 33 ]. this potential protective role of pfm in metabolic disturbances and atherogenicity may be, in part, explained by adiponectin insulin sensitizing effects. in fact, it has been suggested that specifically the thigh subcutaneous at, a major contributor for the circulating adiponectin, could mediate these counteracting effects. although little is known regarding the necessity of making a clear distinction between the metabolic activity and role of the femoral subcutaneous and subfascial fat depots, it was already assumed that subfascial thigh fat, representing the intermuscular (within muscle fibbers) fat deposition, is characterized by a different lipolysis rate and cytokine secretory profile, compared to subcutaneous femoral fat [1, 35 ]. in our study developed in overweight and obese premenopausal women, beside the associations found suggesting that thigh subfascial at may confer a metabolic protection against detrimental ectopic fat storage in the liver, although not significant, a similar trend was observed between both total thigh at mass and subcutaneous at and lsr. in this context, the metabolic differences previously described between both thigh adipose tissue compartments might underlie different mechanisms that could interfere with the relations found in this study. other studies conducted with type 2 dm patients have also reported that thigh sfat is associated with hepatic fat and ir [6, 28 ]. indeed, in a recent study developed with 83 type 2 dm patients, it was observed that fatty liver was inversely related with femoral subfascial at and with visceral adiposity, independently of the effects of vat and bmi. more than interpreting these results as an evidence suggesting a causative role of thigh sfat to fatty liver pathogenesis, the authors have proposed that sfat together with fatty liver are special adiposity depots related with ir pathogenicity in type 2 dm. these results obtained in type 2 dm patients contrast with our observations in overweight and obese premenopausal caucasian women, suggesting that this body composition area warrants more research in order to clarify the possible underlying mechanisms. however, one possible explanation for the differences found between both studies might be related with the fact that the unfavourable metabolic profile normally present in type 2 dm patients contrasts with a relative healthy metabolic pattern found in our sample of overweight and obese premenopausal women (only 9.3% of the women met the atp iii criteria for metabolic syndrome). the role of abdominal obesity on ectopic liver fat storage and the concomitant metabolic abnormalities was already addressed [6, 7, 36, 37 ]. in a study with 144 patients with hepatic steatosis, clinically characterized by hepatocyte fat infiltration and often described as fatty liver, bmi was the unique independent predictor of the steatosis degree. another two studies have also reported that, both in obese patients and in living liver donors, bmi was associated with the steatosis severity. in our study with overweight and obese premenopausal caucasian women, we found that, independently of age, a higher weight, bmi and sagittal diameter (but not wc) were associated with a lower lsr. despite the nonsignificant association verified between lsr and wc when adjusting for age and bmi, in fact one explanation for this observation may be related with the measurement procedures, which may vary according to the method used ; when wc is measured by lohman. contrarily, according to the national health and nutrition examination survey - nhanes iii guidelines, wc is measured immediately above the iliac crests increasing the absolute mean values registered. in our study, despite the nonassociation verified between lsr and wc when adjusting for age and bmi, there is a trend that could be significant if adopted another measurement procedure. the observation made in our study that, for a given wc, increased thigh sfat areas were also significantly related with a higher lsr seems to reinforce the importance of taking into account the wc in this analysis. in addition, another reason to justify the inclusion of wc in these statistical analysis procedures is related with the fact that wc seems to be clinically easer to measure rather than sd and vat (usually implying specific equipment and more complex procedures). on the other hand, in a study with 221 chronic hepatitis c patients, vat, rather than bmi, was a significant predictor of hepatic steatosis. in fact, abdominal obesity markers, such as wc [17, 20 ], whr [20, 42 ], vat [4, 7 ], vat / taat ratio [4, 7 ], and ab sat have been markedly associated with liver fat. in our study, after adjustment for hc, a larger wc was related with liver fat storage. additional adjustments for age, bmi, and thigh sfat revealed that a higher vat area was independently associated with a lower lsr (= 0.250, p 0.05), was, in fact, already suggested in a previous study with type 2 dm patients and in other recent study reporting that surgical vat removal could reverse hepatic ir. the link between abdominal adiposity and liver fat storage may be explained by the fact that ffa are more easily mobilized from visceral at rather than ab sat depots, draining directly into the liver via portal circulation. the increased ffa liver influx may induce hepatic steatosis that might be responsible for other metabolic disturbances, such as increased liver ffa and tg - rich lipoproteins synthesis, adipocyte proliferation failure, and insufficient hepatocyte ffa oxidation [15, 19, 45 ]. in addition, liver lipotoxicity may be accompanied by a low chronic inflammatory state, which can promote a future progression to nonalcoholic steatohepatitis (nash). despite evidence has been demonstrating the vat - derived ffa contribution to these pathophysiologic cascade, a recent overview have also highlighted the role of ffa released from abdominal subcutaneous adipocytes into systemic circulation to these hepatic disturbances. in this context, the results of our study with premenopausal overweight and obese women are consistent with some emerging observations, suggesting that liver fat is strongly associated with abdominal obesity and can also independently reflect an unfavourable metabolic syndrome profile. indeed, we observed that higher insulin, tg, liver transaminases, pai-1, and uric acid concentrations were independently associated with a lower lsr. furthermore, higher tc / hdl - c and ldl - c / hdl - c ratios were also related with lower lsr values. these metabolic markers remained significantly associated with liver fat, independently of vat (data not shown). despite some evidence have been proposing that hepatic fat storage is normally preceded by vat accumulation, our results are consistent with other observations reporting that liver fat remains associated with metabolic syndrome features independently of total and visceral adiposity [22, 47 ]. in this sense, these results suggest that hyperinsulinemia, hypertriglyceridemia and hypercholesterolemia are relevant to the metabolic cascade that mediates liver disturbances in overweight and obese premenopausal women. other studies developed with both insulin - sensitive and insulin - resistant subjects have also reported that liver fat was associated with ir markers and tg concentrations [5, 7 ]. another study developed with type 2 dm patients reported that the presence of fatty liver was associated with a higher degree of ir and dyslipidemia. hepatic steatosis has also been associated with dyslipidemia, hyperinsulinemia, and ir not only in obese subjects but also in lean subjects without glucose intolerance. although the role of diabetes in hepatic steatosis and in its progression to nash still remains unclear, the nhanes - iii reported that simple ir features, such as fasting insulin, hb a1c, and c - peptide concentrations, as well as abdominal obesity markers were independently associated with alt concentrations, the most sensitive indicator of liver cell integrity. in fact, increased liver transaminases concentrations are associated with obesity severity and can also predict the liver injury degree [39, 48 ]. on the other hand, it is noteworthy that hyperinsulinemia seems to play a key role in ffa metabolism and may inhibit hepatocyte mitochondrial beta - oxidation, which can additionally contribute to liver lipotoxicity. the inverse associations of both pai-1 and uric acid with lsr observed in our study emphasize the ectopic liver fat storage relevance to inflammatory and atherothrombotic metabolic syndrome disturbances in overweight and obese premenopausal caucasian women. despite several studies have been demonstrating that metabolic disturbances are associated with ectopic liver fat storage, including ours, desprs. have suggested that it might be the lack of or a dysfunctional subcutaneous adipose tissue that may be responsible for the increase in ectopic fat deposition in the liver, heart, skeletal muscle and pancreas which further increase the cardiovascular disease and type 2 dm risk, rather than the inverse way. in order to address this hypothesis, we investigate in our sample of overweight and obese women the independent contribution of lsr to each one of the metabolic syndrome markers studied. similarly to results previously reported, lsr was associated with fasting insulin (= 0.173, p < 0.001), triglycerides (= 0.205, p < 0.05), tc / hdl - c (= 0.212, p < 0.01), and ldl - c / hdl - c (= 0.469, p < 0.05) ratios, and with both alt (= 0.354, p < 0.05) and ast concentrations after adjustment for age, total fat mass, and vat. the explained variance of lsr to each metabolic risk factor studied varied between 15.5% and 29.2%, showing higher values for tc / hdl - c and ldl - c / hdl - c ratios, liver transaminases, and fasting insulin, respectively. despite the independent associations verified between lsr and both pai-1 (= 0.232, p < 0.01) and uric acid (= 0.200, p < 0.05) concentrations when controlled for age and bmi the similarity of the independent associations verified between lsr, and each one of the metabolic syndrome markers studied in our sample suggests that a biological continuum may underlie the relations making hard to discriminate a cause - consequence interpretation. the role of some adipocytokines, such as leptin and tnf- in hepatic steatosis has also been increasingly studied. recent studies have reported that leptin can mediate lean body tissues protection against lipotoxic damage, being also relevant in lipogenesis blocking, and in muscle insulin - sensitization and fatty acid oxidation enhancement. however, hyperleptinemia, commonly present in visceral obese patients, may aggravate ir and promote liver fat storage. on the other hand, inflammatory cytokines, such as tnf- and il-6, often overexpressed in obese patients or overweight subjects with type 2 dm, have also been associated with liver fat and nash pathogenesis. contrarily to the observed in a previous study with type 2 dm patients, in our study with premenopausal overweight and obese women, both lsr and liver attenuation were not associated with leptin, il-6, tnf-, and with any other specific inflammatory and thrombotic risk factors studied. similar results were obtained when we analysed the independent contribution of lsr to both metabolic markers, after adjusted for age, total fat mass, and vat (data not shown). metabolic profile found in our sample when compared to the unfavourable profile usually found in type 2 dm patients. the ct abdominal and thigh adipose and muscle tissue assessments, as well as the broad list of metabolic features measured and the considerable sample size (n = 140) are some of the strengths of this study. additionally, participants were counseled to refrain from exercise at least 48 hours prior to blood sampling, avoiding metabolic acute exercise interferences. however, there are limitations in our study that warrant mention. first, it is noteworthy that liver attenuation obtained by ct can not quantify absolute liver fat because attenuation of each voxel is a function of its lipid, lean tissue, and water composition. therefore, variations in each one of the components may change the resultant attenuation, adding difficulties in data interpretation. second, despite careful attention in ensuring bloods samples were taken during the follicular stage of menses, we did not control the diet prior blood sampling. since lipid levels of liver and muscle can present slightly acute differences depending on diet, this issue may also slightly influence ct attenuations. third, the low prevalence of women (2.9%) presenting a fatty liver found (as defined by a lsr < 1) in our study is also an important issue to consider since the observations made in our study with overweight and obese premenopausal women can not be extrapolated for obese or diabetic patients presenting a dysmetabolic milieu totally different from relatively healthy metabolic subjects. finally, it is well established that men and women (before menopause) present different body composition patterns. adipose tissue accumulation in overweight and obese men tends to be concentrated in the abdominal area whereas women tend to accumulate fat in gluteal - femoral area. it is also known that these different adipose tissue depots present different metabolic characteristics that might be responsible for the body composition differences observed. in this sense, in our study with overweight and obese premenopausal women, the associations observed can not be extrapolated for male subjects, especially those related with the possible preventive role of thigh sfat to liver lipotoxicity since several metabolic characteristics present in female gluteal - femoral adipocytes seem to be reduced in male adipocytes. in summary, contrarily to previous observations made in obese type 2 dm patients, thigh subfascial adiposity was independent and inversely associated with liver fat in overweight and obese women, suggesting that this thigh at compartment may play a preventive role against detrimental liver ectopic fat storage. conversely, our results emphasize the contribution of a higher bmi and visceral at, especially if associated with hyperinsulinemia, dyslipidemia, and an inflammatory and atherothrombotic profile to the metabolic cascade that dialectically interacts with liver lipotoxicity in overweight and obese premenopausal caucasian women. the authors declare that they have no conflict of interests to report in this research.	abdominal obesity has been associated with liver fat storage. however, the relationships between other body composition depots and metabolic syndrome features with hepatic fat are still unclear. we examined abdominal and thigh adipose tissue (at) compartments associations with liver fat in 140 overweight and obese premenopausal caucasian women. blood lipids and, proinflammatory and atherothrombotic markers associations with hepatic fat were also analyzed. a larger visceral at (vat) was related with liver fat (p < 0.05). contrarily, thigh subfascial at was inversely related to liver fat (p < 0.05). increased fasting insulin, triglycerides, pai-1 concentrations, and a higher total - cholesterol / hdl - cholesterol ratio were also associated with hepatic fat, even after adjustment for vat (p < 0.05). thigh subfascial adiposity was inversely associated with liver fat, suggesting a potential preventive role against ectopic fat storage in overweight and obese women. these results reinforce the contribution of an abdominal obesity phenotype associated with a diabetogenic and atherothrombotic profile to liver lipotoxicity.
rheumatoid arthritis (ra) is a chronic systemic autoimmune disease that about 0.5%-1% of world population are affected by its complaints. the ra affects blood vessels, heart, lungs, muscles, and joints, resulting in bone deformity and osteoporosis. several studies have reported that oxidative stress and production of oxygen - free radicals have important role in ra development and epidemiologic studies have revealed a reverse relationship between dietary intake of antioxidants and ra incidence and due to reduction of intake and absorption of dietary antioxidants in ra patients, the levels of blood antioxidants are decreased too. the antioxidants supplements such as vitamin e, vitamin c, and selenium may control the disturbance of lipid peroxidation and loss of antioxidants markers in patients with ra. vitamin e can interact with nitric oxide and may trigger the gene expression of catalase (cat), glutathione peroxidase (gpx), and superoxide dismutase (sod) enzymes, vitamin c may demolish the peroxides of macrophage activities, zinc may strengthen the immune system, and selenium has an important role as a cofactor of gpx enzyme in reduction of oxidative stress. in past 30 years, controlled trials have conducted to compare the effect of dietary antioxidants and antioxidant - rich diets in controlling of ra clinical outcomes ; however, they could not find a clear statement about antioxidants in ra prevention and treatment due to difference in study period, dose, and different types of antioxidants. regarding to integrity of antioxidant defense system and few clinical studies on combined antioxidant supplements in ra, the aim of this study is to evaluate the effect of combined antioxidant supplements as daily oral capsule on clinical outcomes and antioxidant parameters in female patients with ra for 3 months. a pre - post clinical trial was conducted on female patients with ra for 12 weeks. the study group was selected from 400 registered ra patients in sheikh - al - rais and sina clinics of tabriz university of medical sciences, iran according to inclusion criteria. the inclusion criteria were ra diagnosis by rheumatologist according to american college of rheumatology guidelines-1987, 40 - 60 years old, no change in treatment approach in past 2 months. the exclusion criteria were diabetes mellitus, hypertension, thyroid disorders, liver and kidney failure, cushing syndrome, severe infection, gastric illnesses, smoking, and exposure to daily smoking at home. we followed - up the intake of daily supplement use and type and dose of medications by regular phone calls, so change in type and dose of drugs and antioxidant supplement resulted in omission from study. selenplus capsule (eurovital pharmaceutical company, germany) that contained 50 g selenium, 8 mg zinc, 400 g vitamin a, 125 mg vitamin c, and 40 mg vitamin e. the supplement has been given to patients without trading label. after explanation of the study risks and benefits, written consent form was taken from all subjects. registration code of the local ethics committee of tabriz university of medical sciences is 8912 and registration number in the registration center for clinical trials in iran is irct138901183655n1. in the case of any side effects, patients could leave the study and dose of each antioxidant nutrient was at the recommended dietary allowances amount. at the beginning of the study, accurate clinical examination such as counting the swollen and painful joints was done by a rheumatologist and the validated das-28 form was filled to calculate disease activity index according to following formula. : tyc28 : number of painful joints, syc28 : number of swollen joints, sqrt : square, ln : loge also, dietary intake questionnaire including food frequency questionnaire (ffq) and 24 h recall questionnaire for 3 days (2 working days and 1 weekend) were completed by an expert nutritionist. the ffq was composed of 168 food items that assessed the frequency of the intake in day, month, season, and year semiquantitavely. the recall questionnaire was a dietary detailed form in 6 parts : breakfast, lunch, and dinner with three snacks that filled by face to face interview about the type and amount of the food items. dietary intake of energy, macronutrients, and antioxidant micronutrients analyzed by nutritionist iii software (mam soft research co, usa 1993). the weight of patients was measured by digital scale (after calibration and without shoes), the height was measured by stadiometer (after attachment of 4 points of body to the wall) and the body mass index calculated by quetelet formula. the patients were followed - up every 2 weeks by phone calls and the measurements were repeated after 3 months. five milliliter fasting venous blood samples (8 - 12 h after fasting) were taken from all participants and were kept in -70c freezer (snider 's, germany) until conducting biochemical measurements. biochemical measurements including gpx and sod were measured by spectrophotometric kit (ransel, randox laboratories ltd, uk) and autoanalyzer apparatus (abbott, model alcyon 300, usa) and cat was measured by abei method. tac was determined by spectrophotometric kit (randox tac kit, randox laboratories ltd, uk). serum high - sensitive c - reactive protein (hs - crp) was quantified by photometric kit (pars azmoon company ltd, iran). spss software version 13 (spss for windows, chicago, il, usa) was used for statistical analysis. distribution of data was tested by q - q plot and kolmogorov - smirnov test. for parametric data, paired t - test and in case of nonparametric data, wilcoxon signed rank test was used. the linear regression model was used for adjusting confounding factors such as dietary intake of some selected nutrients. a pre - post clinical trial was conducted on female patients with ra for 12 weeks. the study group was selected from 400 registered ra patients in sheikh - al - rais and sina clinics of tabriz university of medical sciences, iran according to inclusion criteria. the inclusion criteria were ra diagnosis by rheumatologist according to american college of rheumatology guidelines-1987, 40 - 60 years old, no change in treatment approach in past 2 months. the exclusion criteria were diabetes mellitus, hypertension, thyroid disorders, liver and kidney failure, cushing syndrome, severe infection, gastric illnesses, smoking, and exposure to daily smoking at home. we followed - up the intake of daily supplement use and type and dose of medications by regular phone calls, so change in type and dose of drugs and antioxidant supplement resulted in omission from study. selenplus capsule (eurovital pharmaceutical company, germany) that contained 50 g selenium, 8 mg zinc, 400 g vitamin a, 125 mg vitamin c, and 40 mg vitamin e. the supplement has been given to patients without trading label. after explanation of the study risks and benefits, written consent form was taken from all subjects. registration code of the local ethics committee of tabriz university of medical sciences is 8912 and registration number in the registration center for clinical trials in iran is irct138901183655n1. in the case of any side effects, patients could leave the study and dose of each antioxidant nutrient was at the recommended dietary allowances amount. at the beginning of the study, accurate clinical examination such as counting the swollen and painful joints was done by a rheumatologist and the validated das-28 form was filled to calculate disease activity index according to following formula. : tyc28 : number of painful joints, syc28 : number of swollen joints, sqrt : square, ln : loge also, dietary intake questionnaire including food frequency questionnaire (ffq) and 24 h recall questionnaire for 3 days (2 working days and 1 weekend) were completed by an expert nutritionist. the ffq was composed of 168 food items that assessed the frequency of the intake in day, month, season, and year semiquantitavely. the recall questionnaire was a dietary detailed form in 6 parts : breakfast, lunch, and dinner with three snacks that filled by face to face interview about the type and amount of the food items. dietary intake of energy, macronutrients, and antioxidant micronutrients analyzed by nutritionist iii software (mam soft research co, usa 1993). the weight of patients was measured by digital scale (after calibration and without shoes), the height was measured by stadiometer (after attachment of 4 points of body to the wall) and the body mass index calculated by quetelet formula. the patients were followed - up every 2 weeks by phone calls and the measurements were repeated after 3 months. five milliliter fasting venous blood samples (8 - 12 h after fasting) were taken from all participants and were kept in -70c freezer (snider 's, germany) until conducting biochemical measurements. biochemical measurements including gpx and sod were measured by spectrophotometric kit (ransel, randox laboratories ltd, uk) and autoanalyzer apparatus (abbott, model alcyon 300, usa) and cat was measured by abei method. tac was determined by spectrophotometric kit (randox tac kit, randox laboratories ltd, uk). serum high - sensitive c - reactive protein (hs - crp) was quantified by photometric kit (pars azmoon company ltd, iran). spss software version 13 (spss for windows, chicago, il, usa) was used for statistical analysis. distribution of data was tested by q - q plot and kolmogorov - smirnov test. for parametric data, paired t - test and in case of nonparametric data, wilcoxon signed rank test was used. the linear regression model was used for adjusting confounding factors such as dietary intake of some selected nutrients. a total of 39 patients sustained in the study after 12 weeks. the baseline characteristics and dietary intake have been reported in reference no. 20. table 1 indicates basic characteristics of the subjects at the start point of trial and the median of the duration of the disease was 72 months [table 1 ]. the pharmacotherapy regimen did not change during the period of the study in the selected patients and any changes in the dose and type of the drugs resulted in the omission of the study. dietary intake of energy and selected nutrients during 12 weeks of intervention did not differ significantly [table 2 ] and in the linear regression findings, no significant linear relationship between dietary antioxidants values with biochemical indices was observed. the basic characteristics of subjects in the study dietary intake of selected nutrients before and after 12 weeks intervention in our study, das-28 score and serum hs - crp have changed during 12 weeks of intervention (p < 0.01), while the number of swollen and painful joints did not change significantly [table 3 ]. the antioxidant markers of patients including tac, gpx, sod, and cat increased significantly after 12 weeks supplementation (p < 0.01) [table 4 ]. the changes of clinical outcomes in subjects of study before and after 12 weeks intervention the changes of erythrocyte antioxidant parameters in subjects of study before and after 12 weeks intervention in our study, antioxidants supplement for 12 weeks reduced significantly serum hs - crp and das-28 score. the literature review indicates that zinc and selenium supplementation have been used in ra remission and prevention for several years and the similar results of these studies were resulted from multicomponent antioxidants and nutrients as koracevic., showed concurrent supplementation with 37.5 mg vitamin e, 150 mg vitamin c, 1.4 g eicosapentaenoic acid, 0.2 g docosaenoic acid, and 0.5 g gamma linolenic acid could not significantly reduce the number of swollen and painful joints. as the results of alike studies revealed intake of simultaneous antioxidant micronutrients can have a helpful effect against ra progress. in another similar study, 300 mg vitamin c, 5 mg zinc, 25000 international unit vitamin a for 12 weeks reduced the disease activity (p < 0.0001)., study showed that 12 weeks selenium supplementation decreased the number of swollen and painful joints ; however, the results were not statistically significant. some studies have used higher doses of one antioxidant although they did not observe significant improvement in clinical outcomes. it seems that the reason of these findings is due to no increase in antioxidants levels in polymorphonuclear leukocytes and antioxidant defense system in blood cells. as onal., study indicated the pharmacotherapy in patients with ra results in lower levels of zinc and selenium and higher levels of copper in red blood cells, so intake of oral drugs such as corticosteroids and chloroquine elevates the required amount of the antioxidants to suppress inflammatory - like substances. in our study, erythrocyte antioxidant markers including tac, gpx, sod, and cat increased significantly during 12 weeks supplementation due to probable direct effect of oral antioxidants on antioxidants levels. similarly, shinde., have shown that 400 mg vitamin e and 500 mg vitamin c could increase the reduced form of erythrocyte glutathione (p < 0.001), probably because vitamin e is the most important fat - soluble antioxidant and protects the cell membranes against oxidative stress just as vitamin c preserves cytosol and membranes of free radicals activity. furthermore, the results of vanvugt., study indicated that 400 mg alpha - tocopherol, 10 mg lycopene, 5 mg alpha carotene, 10 mg lutein, and 200 mg vitamin c for 12 weeks increased plasma levels of vitamin e, lycopene, lutein, alpha - carotene, and vitamin c and reduced f2-isoprstanes as the oxidative stress marker. shah., illustrated that there is a strong association among the disease activity with antioxidant enzymes markers and they have showed that production of reactive oxygen substances can disturb the immune defense system and modulate inflammation processes to reduce the antioxidant molecules in blood cells. it seems that mixture of antioxidants help to reduce required dose of pain killer drugs and diminish the complaints of disease. since autoimmune diseases such as ra are accompanying with reduction of cellular immune level that results in high coincidence of other chronic diseases, healthy antioxidant - rich diet can improve immune system and compensate the inadequate intake of micronutrients, especially antioxidant - rich supplies of ra patients in northwest of iran. also, consumption of antioxidant micronutrients in the form of dietary items or supplements may be helpful in enhancement of enzymatic and nonenzymatic antioxidants due to strengthen the antioxidant defense system of the body. lack of the control group is the major limitation in this pre - post clinical trial due to limited financial support. one of the strengths is the high response rate of participants (97.5%) and low loss to follow - up during the intervention. also, mild to moderate severity of ra was considered as a criterion of the study and the dietary intake of antioxidants was supposed as confounding factors. the combined antioxidant supplement may improve das-28 score significantly, but it did not change the number of painful and swollen joints statistically significant during 12 weeks, while it could increase tac, gpx, sod, and cat levels. it seems that supplementation with antioxidants may be useful as a complementary treatment in control of clinical outcomes and oxidative stress in patients with ra.	background : this study aims to investigate the effect of antioxidants supplement on clinical outcomes and antioxidant parameters in rheumatoid arthritis (ra).methods : the pre - post study was conducted on 40 female patients with ra in 12 weeks that taken daily one selenplus capsule contained 50 g selenium, 8 mg zinc, 400 g vitamin a, 125 mg vitamin c, and 40 mg vitamin e. about 5 ml venous blood sample was taken from all participants and disease activity score (das) was determined by das-28 formula and high - sensitive c - reactive protein (hs - crp). glutathione peroxidase (gpx) and superoxide dismutase (sod) were measured by spectrophotometric kit and catalase (cat) was measured by abei method. total antioxidant capacity (tac) was determined by spectrophotometric kit. distribution of the variables was assessed using histogram with normal curve as well as kolmogorov - smirnov test and data were analyzed with paired t - test for differences between pre - post data using spss software version 13.5.conclusions:our findings showed that antioxidants may improve disease activity significantly, but it did not affect the number of painful and swollen joints and increased erythrocyte antioxidant levels. antioxidants may be useful for controlling of clinical outcomes and oxidative stress in ra.
it is an aggressive neoplasm that arises from the remnants of the dental lamina and dental organ (odontogenic epithelium) and patients usually present late in life after the tumor has achieved considerable size, to cause facial disfigurement. 70% of ameloblastomas develop in the molar - ramus region of the mandible and are occasionally associated with an unerupted third molar teeth. radiographically an ameloblastoma can be a unilocular or multilocular radiolucent lesion with a honeycomb or soap bubble appearance. there are three forms of ameloblastomas, namely peripheral, unicystic, and multicystic tumors. multicystic ameloblastoma is common and represents 86% of all the cases. our patient was a 42-year - old female with a history of swelling in the left mandibular region that had been present for the four - and - a - half years. physical examination demonstrated a non - tender, left mandibular lesion measuring approximately 15 17 12 cm with normal overlying skin [figures 1 and 2 ]. panoramic and posterior - anterior (pa) skull radiographs revealed a well - defined, large, expansile, multilocular radiolucent lesion, involving the entire hemimandible with medial displacement of the teeth [figures 3 and 4 ]. an axial computed tomography (ct) scan of the mandible showed a well - defined, large, multilocular, expansile radiolucent lesion with erosion, cortical destruction, and thinning of the posterior border of the left ramus of the mandible [figure 5 ]. fine needle aspiration cytology (fnac) of the lesion was performed, which yielded straw - colored fluid. subsequently, the patient underwent incisional biopsy of the left mandibular mass confirming the diagnosis of plexiform ameloblastoma. it was predominantly composed of epithelium arranged as a tangled network of anastomosing strands, enclosing cysts of various sizes, suggestive of plexiform ameloblastoma. after confirmation of the diagnosis, the patient underwent left hemimandibulectomy [figures 6 and 7 ]. the excised mass was sent for histopathology examination, which further confirmed the diagnosis of plexiform ameloblastoma [figure 8 ].. a submental view of the patient demonstrates a left mandibular mass with its lateral and inferior extension. an orthopantomograph (opg) demonstrates a large multilocular, expansile lesion causing thinning of the cortical plates involving the whole of the left hemimandible. a posterior anterior (pa) skull radiograph reveals a multilocular expansile lesion of the left mandible, with medial displacement of the involved teeth. a non - contrast computed tomography (ct) axial view through the left mandible demonstrates multiple locules, with expansion and thinning of both the cortical plates, and perforation along the posterior border of the left ramus. photograph of the gross appearance of the mass, measuring 14 15 10 cm. photomicrograph demonstrates a plexiform ameloblastoma predominantly composed of the epithelium arranged as a tangled network of anastomosing strands, enclosing cysts of various sizes. the radiographic appearance of an ameloblastoma varies from characteristic soap bubble loculations, to unicystic and multicystic radiolucencies, to subtle appearances such as expanded follicles of erupting teeth. the most common location is the posterior mandible associated with impacted teeth and follicular cysts, causing expansion of the cortical plates with scalloped margins and perforations with resorption of the involved teeth in advanced stages. radiographically an ameloblastoma may be mistaken for an odontogenic keratocyst, aneurysmal bone cyst, fibrosarcoma, or a giant cell tumor. ameloblastoma is characterized by the proliferation of epithelial cells arranged on a stroma of conjunctive vascular tissue in locally invading structures that resemble the enamel organ at different stages of differentiation. diverse histological patterns have been described in the literature and include follicular, plexiform, acanthomatous, papilliferous - keratotic, desmoplastic, granular, vascular and those with dentinoid induction. the term plexiform refers to the appearance of anastomosing islands of odontogenic epithelium in contrast to a follicular pattern. the epithelium displayed a stellate, reticulum - type appearance, arranged as a tangled network of anastomosing strands, enclosing cysts of various sizes. in conclusion, although ameloblastoma is one of the most common odontogenic tumors, its final diagnosis can only be confirmed with a histopathological examination. radiographically, ameloblastoma of the mandible can mimic other tumors of the mandible, such as, the odontogenic keratocyst, aneurysmal bone cyst, fibrosarcoma, or a giant cell tumor. a high index of suspicion of ameloblastoma will help triage the patients for further appropriate management.	ameloblastoma is a common and aggressive odontogenic epithelial tumor. it has an aggressive behavior and recurrent course, and is rarely metastatic. ameloblastoma represents 1% of all tumors and cysts that involve the maxillomandibular area and about 10% of the odontogenic tumors. it is primarily seen in adults in the third to fifth decade of life, with equal sex predilection. radiographically, it appears as an expansile radiolucent, with thinned and perforated cortices, and is known to cause root resorption. as it shares common radiographic features with other lesions such as the giant cell tumor, aneurismal bone cyst, and renal cell carcinoma metastasis, a definitive diagnosis can only be made with histopathology. we present an extensive case of plexiform ameloblastoma of the mandible in a 42-year - old female patient.
guillain - barr syndrome (gbs) is an acute immune - mediated polyneuropathy, typically presenting with rapid ascending paralysis caused by an antecedent infection. sarcoidosis is a multi - system noncaseating granulomatous disorder of unknown etiology presenting with bilateral hilar lymphadenopathy, pulmonary reticulonodular opacities, and skin, joint, or ocular lesions. a minority of patients with known sarcoidosis develop neurological complications, making neurosarcoidosis a diagnostic consideration. a 43-year - old australian presented with an acute progressive distal limb weakness and sensory alteration of both hands and feet. this information was subsequently unearthed during his readmission to the intensive care unit (icu). inpatient investigations revealed elevated serum corrected calcium of 2.36 mmol / l (n : 1.10 - 1.30 mmol / l) and an angiotensin - converting enzyme (ace) level of 108 u / l (n : 8 - 52 chest x - rays and high - resolution computed tomography scan of the chest showed extensive fine nodular patterns on both lungs. the patient 's symptoms, apart from the blurred vision, improved with 50 mg of daily oral prednisolone. two weeks postdischarge, he re - presented to the hospital with acute progressive limb weakness which started distally with gradual proximal involvement over 2 days. differential diagnoses for acute flaccid paralysis relevant to an intensive care unit patient neurological examination of the upper and lower limbs showed global areflexia. magnetic resonance imaging (mri) of the brain and spine demonstrated abnormal enhancement of the cauda equina nerve roots and patchy white matter signal change representing inflammatory demyelination extending up to the t3 level of the thoracic cord, preferentially involving the ventral nerve roots [figures 1 and 2 ]. cerebrospinal fluid (csf) findings showed acellular fluid with a mildly elevated protein level of 0.67 magnetic resonance imaging of the spine showing high signal and linear contrast enhancement of the cauda equina magnetic resonance imaging of the spine showing enhancement preferentially of the ventral nerve roots based on these findings, a diagnosis of gbs was made, and the patient was started on a 5-day course of immunoglobulin. over the next 24 h, he clinically deteriorated to mrc power 0/5 in all his limbs, with respiratory failure requiring ventilatory support. his stay was complicated by ventilator - associated pneumonia and he underwent a tracheostomy. as he failed to improve after a full dose of immunoglobulin, plasmapheresis was initiated. sarcoidosis is a multisystem inflammatory disorder of unexplained etiology. in a prospective study from australia, allen. neurological manifestations in a patient with known sarcoidosis should always prompt a possible diagnosis of neurosarcoidosis. the most common brain mri finding of neurosarcoidosis is a basilar leptomeningeal involvement (in about 30 - 40%), which is usually seen as a thickening and diffuse or nodular enhancement. nonspecific anomalies that support a diagnosis of neurosarcoidosis are elevated ace, pleocytosis, reduced or normal glucose, high protein count (> 0.5 g / l), elevated beta-2 microglobulin and increased igg index with possible oligoclonal bands. evidence of patchy demyelination with conduction block has been seen, which is most likely as a result of mechanical neural compression by granulomata. a risk estimate of one additional gbs case per million people vaccinated is found in the advisory committee on immunization practices recommendation and the vaccine information statement for influenza vaccine. the period between vaccination and first symptom onset ranged could potentially range from as short as 3 - 5 days to 6 - 18 weeks, and up to a few months and even years. this correlated with the peak incidence of vaccine - associated neuromuscular weakness as differential diagnoses, even though it was hard to prove that the vaccine had definitely caused the axonal neuropathy. the other speculation with regards to the vaccination as a precipitant of the inflammatory polyradiculoneuropathy was the axonal pattern on the nerve conduction study as opposed to the typical demyelinating pattern with conduction blockade and reduced amplitude which occurs with aidp. furthermore, the h response and f wave were not particularly abnormal keeping in with a typical gbs, which happens following an infectious prodrome. neuroimaging studies and csf analysis results were also consistent with gbs as opposed to neurosarcoidosis. peripheral nerve conduction studies hold a prognostic value and frequently show classical demyelination but can show axonopathy and inexcitability in certain subgroups of gbs. mri findings of gbs report marked enhancement of the thickened nerve roots in the conus medullaris and cauda equina, with no abnormalities on precontrast images. these mri features were in contradistinction to the pachymenigeal and leptomeningeal enhancement which is typical of neurosarcoidosis along with involvement of the perivascular spaces, the circle of willis, and the cranial nerves. corticosteroids are the first - line agents while other immunomodulatory and biological agents can be used. this is in stark contrast to the treatment of gbs in which steroid therapy has no proven benefit. starting with ivig or plasmapheresis would be a reasonable option in these patients, especially given the concern about a poor response with steroids for typical gbs. nevertheless, if there is an unexpectedly high pleocytosis in csf followed by additional confirmation of sarcoidosis, following up with further steroid treatment for neurosarcoidosis is highly recommended. the authors are happy to report this interesting case illustrating the neurological complications of sarcoidosis in a man with rapid onset peripheral neuropathy, which prompted further investigations for neurosarcoidosis and the eventual results that conversely supported a diagnosis of acute gbs. this case also illustrates the importance of accurate history taking in patients with extreme complexity, especially in an icu environment, wherein the art of history taking is regrettably sidelined due to the vagaries of time, conflicting commitments and the absence of a reliable historian. our report also highlights the temporal association between influenza vaccine and gbs, even though causality has not been proven.	guillain barr syndrome (gbs) is an acute demyelinating polyneuropathy, usually evoked by antecedent infection. sarcoidosis is a multisystem chronic granulomatous disorder with neurological involvement occurring in a minority. we present a case of a 43-year - old caucasian man who presented with acute ascending polyradiculoneuropathy with a recent diagnosis of pulmonary sarcoidosis. the absence of acute flaccid paralysis excluded a clinical diagnosis of gbs in the first instance. subsequently, a rapid onset of proximal weakness with multi - organ failure led to the diagnosis of gbs, which necessitated intravenous immunoglobulin and plasmapheresis to which the patient responded adequately, and he was subsequently discharged home. neurosarcoidosis often masquerades as other disorders, leading to a diagnostic dilemma ; also, the occurrence of a gbs - like clinical phenotype secondary to neurosarcoidosis may make diagnosing coexisting gbs a therapeutic challenge. this article not only serves to exemplify the rare association of neurosarcoidosis with gbs but also highlights the need for a high index of clinical suspicion for gbs and accurate history taking in any patient who may present with rapidly progressing weakness to an intensive care unit.
human endometrium undergoes regular cycles of growth and breakdown and has long been recognized as one of a few tissues where significant angiogenesis occurs on a physiological basis (1). these vascular changes are thought to be mediated by the vascular endothelial growth factor (vegf) and its specific receptors (2, 3). six members of the vegf family, vegf - a, vegf - b, vegf - c, vegf - d, vegf - e and placental growth factor (plgf) have been identified to date. these growth factors exert their activity through activation of three types of receptors : vegfr-1 (flt-1), vegfr-2 (flk-1/kdr), and vegfr-3 (flt-4) (1, 4, 5). of those, vegfr-2 (flk-1) seems to be mainly involved in the formation of primitive endothelial tubular structures after binding with vegf - a and vegf - c (6). human endometrium expresses all of the splice variants of vegf (7). as an estrogen - responsive angiogenic factor that varies throughout the menstrual cycle, vegf may be important in both physiological and pathological angiogenesis of human endometrium (8). vegf is involved in several functions of the female reproductive system, such as ovulation, periodical changes of the endometrium, embryo implantation and development. in addition, dysregulation of vegf seems to play a key role in conditions such as preeclampsia and fetal intrauterine growth restriction (9). previous studies have shown that vegf and its receptors are expressed in the mouse and rabbit endometrium and probably participate in the increased angiogenesis and vascular permeability necessary for implantation (10, 11). deficient expression of vegf and its receptors in mice result in poor development of vascular network in the endometrium leading to implantation failures and abortions (12). expression of vegf and its receptors is significantly increased during the post - ovulation and around the peri - implantation period. it appears that the expression of vegf is highly regulated in a temporal and spatial manner at the early stage of implantation (1). knowledge on the expression and regulation of vegf and its receptors in human endometrium during menstrual cycle is still limited and the results are controversial. a previous study reported that the expression of vegf receptors (flt-1 and flk-1/kdr) is temporarily regulated according to the phase of the cycle and these changes are responsible for vegf actions on endometrial vascular growth and permeability (2). reported that vegf, flk-1 and flt-1 were differentially expressed in the endometrial epithelium and stroma during the proliferative and secretory phases (3). vegf receptors exhibited the strongest expression during the beginning of the secretory phase, coinciding with the developing endometrial edema and formation of a complex subepithelial capillary plexus (13, 14). the aim of this study was to investigate the expression pattern of vegf and flk-1 in three components (luminal epithelium, glandular epithelium and stroma) of human endometrium during different phases of menstrual cycle using tissue microarray (tma) analysis. to date, the expression pattern of vegf and flk-1 during the menstrual cycle has not been investigated thoroughly in such a manner. the study was performed at the johns hopkins university school of medicine and the institutional review board of johns hopkins hospital approved this study. archived paraffin - embedded endometrial samples from 60 normally cycling women (age 23 - 39) were obtained from the department of pathology of the johns hopkins hospital from july 2001 to june 2003. all patients had regular menstrual cycles ranging in length from 28 to 30 days and had not received exogenous hormonal therapy for at least 2 months before the procedure. women with sexually transmitted diseases, pelvic inflammation diseases, systemic diseases, and any reproductive tract pathology were excluded from the study as well as those with bmi greater than 28. samples with evidence of endometritis, endometrial polyp, endometrial hyperplasia, or other pathologies were also excluded. the samples were classified histologically according to the criteria of noyes (15). to allow for a high throughput tissue analysis on paraffin - embedded samples, tissue chip approach was used. tissue microarray (tma) is a method of harvesting small - core biopsies from a range of standard histological sections and placing them in an array on a recipient paraffin block in such a manner that hundreds of specimens can be analyzed simultaneously. this technique provides a highly efficient, reliable, and high - throughput mechanism for evaluation of protein expression in large cohorts. therefore, tma allows a rapid and comprehensive characterization of biomarkers of interest (16). in this study, tma slides were assembled from formalin - fixed, paraffin - embedded endometrial samples at cores of 1.5 mm in diameter for each core and three representative punches from each specimen. each tissue core was sectioned in 5 m thickness and affixed to the glass slides. examples of tissue microarray slides immunostained with vegf and flk-1 antibodies tissue sections were dewaxed through descending grades of ethanol to distilled water, and pretreated with citra buffer (vector h3300, vector laboratories, burlingame, ca) in a steamer (ha900 ; black & decker, hampstead, md) at 90c for 20 min. immunohisto - chemical staining was then performed using monoclonal antibodies of anti mouse vegf (sc-7269) and anti - human flk-1(sc-6251) (santa cruz biotechnology inc, ca). binding of vegf or flk-1 was detected by a biotinylated rabbit antimouse secondary antibody, followed by hrp conjugated avidin - biotin perioxidase. for the negative control, additional sections were stained with an antihuman microthalmia transcription factor (mitf) antibody (clone d5 ; department of pathology, johns hopkins medical institutes, baltimore, md). the primary antibody was detected by using a ventana dab detection kit (ventana - biotek solutions, tucson, az). the stained tma slides were evaluated using a light microscope (olympus, ch-2, hitech instruments, inc. the intensity of staining of the antibody was then analyzed by hscore, a semi - quantitative method, which has been shown to have a low intra - observer and inter - observer variability. the hscore was calculated using the following equation : hscore = s pi (i + 1), where i is the intensity of the stained epithelium (1 = weak, 2= moderate and 3 = strong), and pi is the percentage of stained epithelial cells for each intensity varying from 0 to 100%. a commercially available statistical package (spss13.0, spss inc., non - parametric kruskal - wallis test and one - way analysis of variance (anova) were used to compare differences in the hscores between five phases of the menstrual cycle. when there was a significant difference in the main effect, scheff 's posthoc test was performed to identify the significant differences between the phases. archived paraffin - embedded endometrial samples from 60 normally cycling women (age 23 - 39) were obtained from the department of pathology of the johns hopkins hospital from july 2001 to june 2003. all patients had regular menstrual cycles ranging in length from 28 to 30 days and had not received exogenous hormonal therapy for at least 2 months before the procedure. women with sexually transmitted diseases, pelvic inflammation diseases, systemic diseases, and any reproductive tract pathology were excluded from the study as well as those with bmi greater than 28. samples with evidence of endometritis, endometrial polyp, endometrial hyperplasia, or other pathologies were also excluded. to allow for a high throughput tissue analysis on paraffin - embedded samples, tissue chip approach was used. tissue microarray (tma) is a method of harvesting small - core biopsies from a range of standard histological sections and placing them in an array on a recipient paraffin block in such a manner that hundreds of specimens can be analyzed simultaneously. this technique provides a highly efficient, reliable, and high - throughput mechanism for evaluation of protein expression in large cohorts. therefore, tma allows a rapid and comprehensive characterization of biomarkers of interest (16). in this study, tma slides were assembled from formalin - fixed, paraffin - embedded endometrial samples at cores of 1.5 mm in diameter for each core and three representative punches from each specimen. each tissue core was sectioned in 5 m thickness and affixed to the glass slides. tissue sections were dewaxed through descending grades of ethanol to distilled water, and pretreated with citra buffer (vector h3300, vector laboratories, burlingame, ca) in a steamer (ha900 ; black & decker, hampstead, md) at 90c for 20 min. immunohisto - chemical staining was then performed using monoclonal antibodies of anti mouse vegf (sc-7269) and anti - human flk-1(sc-6251) (santa cruz biotechnology inc, ca). binding of vegf or flk-1 was detected by a biotinylated rabbit antimouse secondary antibody, followed by hrp conjugated avidin - biotin perioxidase. for the negative control, additional sections were stained with an antihuman microthalmia transcription factor (mitf) antibody (clone d5 ; department of pathology, johns hopkins medical institutes, baltimore, md). the primary antibody was detected by using a ventana dab detection kit (ventana - biotek solutions, tucson, az). the stained tma slides were evaluated using a light microscope (olympus, ch-2, hitech instruments, inc. the intensity of staining of the antibody was then analyzed by hscore, a semi - quantitative method, which has been shown to have a low intra - observer and inter - observer variability. the hscore was calculated using the following equation : hscore = s pi (i + 1), where i is the intensity of the stained epithelium (1 = weak, 2= moderate and 3 = strong), and pi is the percentage of stained epithelial cells for each intensity varying from 0 to 100%. non - parametric kruskal - wallis test and one - way analysis of variance (anova) were used to compare differences in the hscores between five phases of the menstrual cycle. when there was a significant difference in the main effect, scheff 's posthoc test was performed to identify the significant differences between the phases. out of the 60 samples selected in the study, 23 were obtained from patients who had undergone hysterectomy for uterine leiomyoma and the remaining 37 were biopsies as a part of infertility evaluation. the samples were divided into five groups according to the day of sampling : proliferative (days 7 - 12, n = 14), peri - ovulatory (days 13 - 15, n = 9), early - secretory (days 16 - 18, n = 12), mid - secretory (days 19 - 21, n = 11) and late - secretory (days 24 - 26, n = 14) phases. mean age and bmi of the patients among groups were similar (p > 0.05). the demographic variables of the patients (meanse) vegf and flk-1 were immunolocalized in the luminal epithelium, glandular epithelium and stroma of the endometrium at various phases of the menstrual cycle (figures 2 and 3, a - e). no immunoreactivity was detected when a monoclonal antibody mitf was used as the primary antibody in the negative control staining (figures 2 and 3, f). endometrial tissues at the a : proliferative phase (days 7 - 12) ; b : peri - ovulatory phase (days 13 - 15) ; c : early - secretory phase (days 16 - 18) ; d : mid - secretory phase (days 19 - 21) and e : late - secretory phase (days 24 - 26). endometrial tissues at the a : proliferative phase (days 7 - 12) ; b : peri - ovulatory phase (days 13 - 15) ; c : early - secretory phase (days 16 - 18) d : mid - secretory phase (days 19 - 21) and e : late - secretory phase (days 24 - 26). the red arrows indicate positive immunostaining table 2 summarizes the hscores of vegf and flk-1 in the endometrium during the menstrual cycle. for different locations but the same phase of the cycle, the expression of vegf showed no statistically significant difference among the different compartments although the immunoreactivity was overall stronger in the luminal and glandular epithelium than that in the stroma (figures 1a - e, table 2). within the luminal epithelium, the expression of vegf showed a trend of down regulation from the proliferative phase to late - secretory phase. in the glandular epithelium, a stronger staining intensity was observed at the early - secretory phase, and then it trended down towards late - secretory phase. reactivity of vegf varied significantly in the stroma between the phases of the cycle (p < 0.05), as tested by one - way anova. the expression and distribution of vegf and flk-1 at the various phases of the normal cycle significant differences were observed in the stroma among the phases (p < 0.05) significant differences at the peri - ovulatory phase were observed between luminal epithelium and stroma and between glandular epithelium and stroma (p < 0.05) significant differences at the early - secretory phase were observed between luminal epithelium and stroma and between glandular epithelium and stroma (p < 0.05) significant differences at the latesecretory phase were observed between luminal epithelium and stroma and between glandular epithelium and stroma (p < 0.05) expression of flk-1 showed distinct patterns and changes during the menstrual cycle (figures 2a - e, table 2). a stronger staining intensity was detected in the luminal and glandular epitheliums in all phases of the menstrual cycle, while the greatest intensity was seen during late - secretory phase in the three components of the endometrium. the expression of flk-1 was weaker in the stroma as compared to the luminal and glandular epithelium at the peri - ovulatory, early - secretory and late - secretory phases (p < 0.05) (table 2). the human endometrium is a dynamic tissue that undergoes cyclic proliferation and differentiation controlled by a sequential and carefully timed interplay of various hormonal and environmental changes. the physiological changes in the endometrium during the menstrual cycle are associated with profound angiogenesis. vegf stimulates endothelial cell proliferation, permeability, migration and assembly into capillary tubes (17). in a previous study on expression of vegf and its receptors in human endometrium during menstrual cycle and in early pregnancy, sugino. reported that vegf and its receptor flk-1 (kdr) were expressed in both glandular epithelial and stoma cells during the mid - secretory phase as well as in decidual cells of early pregnancy (3). with a limited number of cases, the study failed to show the expression of vegf and its receptors in luminal epithelium. in the present study, an attempt was made to investigate the temporal and spatial distribution of vegf and its receptor flk-1 in the luminal epithelium, the glandular epithelium and the stroma of the endometrium throughout the menstrual cycle using a high throughput tissue microarray analysis, which can efficiently investigate the gene expression patterns in a sizable number of samples simultaneously. the expression of vegf and flk-1 were identified in all of the three components of the endometrium. the immunoreactivity of vegf in the luminal and glandular layers of the endometrium was overall stronger than that in the stroma where the vegf expression differed significantly between the phases of the cycle. while the expression of vegf in the luminal epithelium showed a trend of down regulation from the proliferative phase to late - secretory phase, stronger intensity of flk-1 was detected in the luminal and glandular epitheliums in all phases of the menstrual cycle with greatest intensity during late - secretory phase. torry. (18) detected strong vascular endothelial growth factor immunoreactivity in the glandular epithelial cells of the secretory endometrium with no discernible immunoreactivity in stroma cells. observation by naresh 's group (19) showed that glandular expression of vegf was higher as compared to stromal compartment. using immunocytochemistry and computerized image analysis of the endometrial functionalis, showed that the number of capillaries immunostained for flk-1/kdr was maximal during the proliferative phase, while co - expression of flk-1/kdr and flt-1 peaked during the mid - secretory period, in synchrony with the characteristic increase of endometrial microvascular density and permeability (2). recent studies have reported that the role of vegf in the early angiogenic processes is associated with postmenstrual regeneration of the endometrium (20). vegf is essential for the rapid burst of angiogenesis that occurs during postmenstrual repair and in the early proliferative phases in the primate endometrium, and further plays a role in re - epithelialization of the endometrium. the temporal and spatial distribution of vegf and its receptor would thus appear to be related to the process of menstruation. the expression of vegf in the human endometrium is reportedly regulated by ovarian steroid hormones. estrogen has been found to induce vegf expression in the stromal layer of the uterus in rodents, as well as in cultured human uterine stromal cells (21). ovarian sex hormones may also regulate vegf activities through modulating the expression and function of the vegf receptors. administration of estrogen plus progesterone enhanced the levels of expression for vegf and its receptors (3). in isolated endometrial stromal cells from proliferative phase endometrium, incubated with estrogen and medroxyprogesterone acetate for 18 days, expression of vegf and kdr mrnas was significantly increased, whereas flt-1 mrna expression was not affected (3). in the present study, the expression of flk-1 predominantly increased at the peri - ovulatory and early - secretory phases in the glandular epithelium of the endometrium, when estradiol levels reached their peak during the natural cycle, suggesting that flk-1 expression was closely associated with cycling steroid hormone changes. a recent study by herve. showed that 17 beta - estradiol up - regulates flk-1/kdr expression in vivo in endothelial cells mainly through the modulation of vegf by a paracrine mechanism (22). the findings of the present study on a high level of flk-1 expression in the luminal and glandular epithelium (table 2) may suggest the role of flk-1 in the preparation of the endometrium for vascularization and implantation. a recent review by okada. summarized regulations of decidualization and angiogenesis in the human endometrium (17). following treatment with estrogen, increase in vegf and decrease in svegfr-1 production, and consequential increase in vegf / svegfr-1 ratio appears to be a sustained and ongoing process designed to promote growth and development of the endometrium during the advancing stages of the menstrual cycle at the local level. in addition, co - treatment with the progesterone receptor antagonist ru-486 reverses this inhibition of estrogen - stimulated vegf, suggesting a pathway by which progestins may reduce the production of these factors through the progesterone receptor (23). progestins are known to initiate down regulation of the estrogen receptor in the human endometrium in vivo as well as in vitro, and therefore the inhibition of vegf may be caused by the decrease in estrogen receptor levels (23). in our study, variation in the expression of vegf in stroma and differential expression of flk-1 in different compartments during menstrual cycle may reflect the balance of steroid hormone 's effect in the dynamic changes of the cycle. the significance of differential expression of vegf and its receptor at given phases and locations of the endometrium remain to be further studied and elucidated. it was recognized that one of the limitations of this study is lacking the data of measurement for hormones which affect the menstrual cycle. nevertheless, the strengths of this study include its relatively large sample size, physiological status of the specimens, reliable high throughput semi - quantitative technology, and thorough measurements on the levels of vegf and flk-1 expression in the human endometrial tissue in a temporal and spatial manner, which has not been completely investigated in the previous studies. the results reported here may reflect the aspects of endometrial process of angiogenesis during menstrual cycle. documentation of this study would be a valuable addition to our current understanding and to the bank of literature for the expression and distribution of vegf and its receptors in human endometrium in physiological conditions. in summary, it was demonstrated that vegf and flk-1 underwent differential expression in the three components of human endometrium at various phases throughout the normal menstrual cycle. the results suggest functional role(s) of these factors in the cycling changes and remodeling process of the endometrium.	backgroundthe vegf is essential in the process of tissue remodeling and angiogenesis. limited data is available on the expression and regulation of vegf and its receptors in the human endometrium. the aim of this study was evaluation of expression patterns of vegf and flk-1 in human endometrium during the menstrual cycle.methodssixty paraffin - embedded blocks of endometrial tissues from the patients with normal menstrual cycles were obtained. tissue samples were assembled into tissue microarray slides and classified by histological dating into five phases : the proliferative (n = 14), peri - ovulatory (n = 9), early - secretory (n = 12), mid - secretory (n = 11) and late - secretory (n = 14) phases. immunohistochemical staining was performed using vegf or flk-1 monoclonal antibodies. the intensity of immunostaining was evaluated by the semi - quantitative scoring method (hscore). kruskal - wallis one - way analysis of variance and scheff 's post - hoc test were used for statistical analysis. a p - value of < 0.05 was considered statistically significant.resultsvegf and flk-1 were expressed in the three components of the endometrium at various phases of the menstrual cycle. in the stroma, the expression of vegf varied among the phases (p < 0.05). the expression of flk-1 in the luminal and glandular epithelium revealed stronger intensity of immunostaining as compared with the stroma at the different phases (p < 0.05). the level of flk-1 expression also showed significant differences among the phases in the glandular epithelium with greatest expression at late - secretory phase (p < 0.05).conclusiontemporal and spatial distribution of vegf and flk-1 expression in the three components of human endometrium during menstrual cycle suggests the functional role of angiogenesis in the remodeling process of endometrial tissue.
dermatophytes comprise a highly specialized group of pathogenic fungi that infect keratinized tissues (skin, hair, and nails) of humans and animals, resulting in dermatophytoses, also referred to as tinea infections. taxonomically, dermatophytes are classified into three genera : epidermophyton, microsporum, and trichophyton, with the latter being the most complex, containing more than 15 species and several different variants within the species t. mentagrophytes. the most frequently observed dermatophyte species worldwide is t. rubrum, whose infections usually manifest as tinea pedis and tinea unguium (onychomycosis). the prevalence of t. rubrum as a causative agent of onychomycosis is particularly high and exceeds 90% in europe. the identification of t. rubrum by means of conventional laboratory methods may not always be easy or straightforward, since t. rubrum exhibits substantial phenotypic variability. contrastingly, a high degree of homogeneity of the t. rubrum genome, as revealed by using several anonymous molecular markers, significantly impedes discrimination at the strain level [3, 4 ]. yet, as new molecular typing methods are becoming increasingly available, species determination and strain typing are still being improved. the aim of this study was to apply some of the recently devised dna fingerprinting methods to the identification and strain differentiation of t. rubrum. the study included 57 isolates of t. rubrum recovered from as many dermatological patients from lower silesia, poland (40), krakw, poland (8), and tbingen, germany (9). clinically, onychomycosis showed the highest number of cases (40 patients ; 70.2% of all patients), followed by tinea pedis (12 ; 21%), tinea corporis (2 ; 3.5%), tinea cruris (2 ; 3.5%), and tinea manuum (1 ; 1.8%). the isolates were primarily identified as t. rubrum on the basis of their phenotypic characteristics, such as colony surface texture, reverse pigmentation, the ability of micro- and macroconidia formation, and urease activity, assessed by standard mycological procedures. the identification of t. rubrum was achieved by restriction fragment length polymorphism (rflp) analysis of the internal transcribed spacer (its) region of rdna, as previously described. the amplified products were digested with mvni, hinfi, and, where necessary, with mvai restriction endonucleases (roche), at a 2-h incubation at 37c. the resulting restriction fragments were separated electrophoretically on 8% polyacrylamide gels and visualized under uv light after ethidium bromide (et - br) staining. two primers, designated 1 and 6, as devised by baeza and mendes - giannini, were used in two separate randomly amplified polymorphic dna (rapd) assays. amplification products were resolved by electrophoresis using 1.5% agarose gels and were photographed under uv light after et - br staining. the gel images were analyzed by the gel doc system and quantity one (bio - rad, usa) software. a dendrogram of the rapd profiles obtained with primer 1 was constructed using dice s coefficient of similarity and the unweighted pair - group arithmetic averaging (upgma) clustering method. of the 57 clinical isolates assessed in this study and recognized as t. rubrum by conventional identification methods, 55 (96.5%) were further confirmed as t. rubrum by means of pcr - rflp analysis. using mvni, all of the isolates except for one (841/05) yielded restriction patterns consistent with that of trichophyton taxon. using hinfi, 55 (96.5%) one isolate (841/05) could not be assigned to any of the dermatophyte species, distinguishable by their unique rflp profiles obtained with mvni, hinfi, or mvai enzymes. among the 55 t. rubrum isolates, a total of 40 distinct patterns (a an) were obtained by rapd with primer 1. the four most prevalent patterns, designated as t, aj, ae, and an, contained five, four, three, and three isolates, respectively. four patterns (c, af, ag, and al) contained two isolates each, and the remaining 34 patterns were represented by single isolates. the rapd with primer 6 generated five different profiles in t. rubrum isolates (a e) and two additional profiles in non - t. rubrum isolates (designated as f and g for isolate nos. b, found in 23 isolates, followed by profiles designated as a and c, represented by 19 and 11 isolates, respectively. patterns designated as d and e were each identified for one isolate. the combination of the profiles obtained with both primers resulted in 47 different genotypes for the t. rubrum isolates. six of those genotypes were common to three (type t - a and type aj - a) or two (types c - a, ae - b, af - b, and an - a) isolates, whereas the remaining 41 genotypes were unique, which are represented by single isolates only. the distribution of the genotypes varied among the three geographical regions from which the isolates originated. of the 40 rapd genotypes produced with primer 1, each twenty - six genotypes occurred in isolates from lower silesia, eight genotypes were observed in isolates from krakw, and six in isolates from tbingen, germany. regarding the genotypes obtained by rapd with primer 6, genotypes b and c were found in isolates from all three regions studied, and genotype a was observed in the lower silesia and tbingen isolates, but not in the krakw isolates. c were the most prevalent in single regions, i.e., in lower silesia (44.7% of isolates), krakw (62.5%), and tbingen (44.4%), respectively. a dendrogram based on the rapd profiles with primer 1 allowed the separation of the t. rubrum isolates into genetic similarity groups (clusters). a total of 48 isolates could be allocated into six main clusters (i vi), with the similarity index between the isolates within the cluster being 80% or higher (fig. 1). the largest cluster (vi) comprised 21 isolates, for which 11 different patterns were obtained. clusters iv and v consisted of eight isolates each, being representative of either four (cluster iv) or eight (cluster v) distinct patterns. the numbers of isolates (and corresponding patterns) belonging to the remaining clusters were 3 (2 patterns) for cluster i, 5 (5) for cluster ii, and 3 (3) for cluster iii. 1dendrogram showing genetic relationships among 55 trichophyton rubrum strains inferred from the randomly amplified polymorphic dna (rapd) patterns generated by using primer 1 dendrogram showing genetic relationships among 55 trichophyton rubrum strains inferred from the randomly amplified polymorphic dna (rapd) patterns generated by using primer 1 the traditional, culture - based methods of identifying dermatophytes are cumbersome, laborious, and often inconclusive, due to fungal phenotypic variability and pleomorphism [1, 8 ]. however, the advent of molecular biology tools has enabled the development of new molecular approaches to the diagnosis of dermatophyte infections., relies upon rflp analysis of pcr - amplified its regions of the rdna gene complex. the only modification to the original procedure was the use of mvni and hinfi instead of mvai. whereas digestion with mvni allows discrimination between the three main dermatophyte genera (trichophyton, microsporum, and epidermophyton), the hinfi digestion results in species - specific restriction profiles. by using those two restriction enzymes, we wanted to verify their usefulness in the molecular identification of dermatophyte species, and t. rubrum in particular. the results from this study showed high concordance between conventional and molecular techniques for identifying t. rubrum. rubrum isolates : one was identified as t. tonsurans (upon restriction analysis with mvai) and the other was concluded to be a non - dermatophyte fungus. a possible explanation for the latter may relate to the laboratory contamination of the specimen and/or culture. it is also noteworthy that the application of the pcr - rflp analysis, as in this study, does not allow to distinguish between t. rubrum and t. soudanense. the distinction between the two species is only possible with methods that target single nucleotide polymorphisms (snps) within the its regions of rdna. given, however, that t. rubrum differs substantially from t. soudanense in terms of phenotypic properties and geographical distribution (the former is cosmopolitan, while the latter is restricted mainly to the sub - saharan part of africa), the assignment to the t. rubrum species provided by pcr - rflp seems unambiguous. differentiation of t. rubrum at the strain level has been attempted using a number of genotyping methods, though with unsatisfactory results [3, 13, 14 ]. have recently reported on intraspecific variability within t. rubrum by pcr - amplifying two tandemly repetitive subelements (trss), located in the non - transcribed spacer (nts) region of the rrna gene cluster. more recently, the rapd analysis performed by baeza. with two decameric primers, designated 1 and 6, has been shown to produce a high degree of interstrain polymorphism [7, 16 ]. the rapd analysis with primers 1 and 6 was also applied in the present study, and this choice was motivated by a high discriminatory potential of the method, higher than that of the trs typing system. based on the combined rapd profiles, a total of 47 distinct genotypes were obtained. hence, the overall genetic diversity rate (gdr), calculated as the number of different genotypes divided by the number of isolates, was 85.4%. it is noteworthy that most of the polymorphism was generated by rapd with primer 1. it yielded 40 profiles, whereas rapd with primer 6 resulted in only five profiles (gdrs of 72.7% and 9.1%, respectively). in the first study that used primers 1 and 6, among ten clinical isolates of t. rubrum, five molecular patterns were observed for each primer. in a subsequent study including 67 t. rubrum isolates, a total of 12 and 11 individual patterns were obtained by rapd with primers 1 and 6, respectively (gdrs of 17.9% and 16.4%, respectively). another study that investigated the intraspecific diversity of t. rubrum isolates originating from japan and china revealed a considerable tightness of the population structure. all 150 isolates tested were split into only 19 fingerprinting genotypes, based on the combined rapd analyses with primers 1 and 6 (gdr of 12.7%). much higher genotypic variability with the same primers was shown in a recent study of santos.. nineteen different molecular profiles were configured for 52 t. rubrum isolates when each of the primers was used independently, resulting in a gdr of 36.5%. it was speculated by the authors that the greater genetic diversity revealed in their study might result from having used strains exclusively from patients with onychomycosis. this condition, with its frequent chronicity, has been associated with mixed infections by multiple t. rubrum genotypes. collectively, the results of the studies cited above differ in terms of genetic polymorphism achieved by rapd with both of the primers. the polymorphism obtained in our study was exceptionally high, and this may relate to the specific, genetic structure of polish (and german) isolates, which is different from that of the so - far - analyzed t. rubrum populations. the polymorphism obtained by rapd with primer 1 was further investigated by a dendrogram analysis derived from the similarity coefficients between the rapd patterns. although the similarity coefficient value for the entire t. rubrum population studied was calculated to be 52%, a vast majority of isolates (87.3%) were distributed into six clusters, within which all of the isolates shared at least 80% similarity. this finding, considering that the similarity coefficient value of 0.80.99 is assumed to represent genetically related isolates, indicates that the clustered isolates might have originated from the same strain that had undergone microevolutionary changes. thus, the high polymorphism resolved by rapd may relate to the reproducibility of the rapd technique itself. indeed, rapd assays often suffer from poor reproducibility and variations in the amplification patterns between isolates may be caused by even the slightest changes in the pcr reaction conditions. this explains the reluctance on the part of researchers to use the rapd method and their search for newer molecular tools with a better diagnostic performance. one such promising alternative method is multilocus microsatellite typing (mlmt), which has recently been developed by grser.. finally, the fact that every genetic cluster identified in rapd with primer 1 was restricted to a single geographical locale, together with marked differences in the frequencies of the three most prevalent genotypes produced from rapd with primer 6, may suggest that certain genotypes exhibit regional and/or geographical affinities. however, since this study was carried out on a relatively small sample of isolates, the geographical specificity of the genotypes would need to be confirmed by further research involving a larger population. interestingly, the geographical differentiation of t. rubrum populations has, so far, been revealed only by using the mlmt methodology [4, 21 ]. in conclusion, the results from this study demonstrated the usefulness of pcr - rflp in the rapid identification of t. rubrum, yet the insufficient suitability of the rapd analysis for t. rubrum strain differentiation, due to its poor reproducibility.	trichophyton rubrum represents the most frequently isolated causative agent of superficial dermatophyte infections. several genotyping methods have recently been introduced to improve the delineation between pathogenic fungi at both the species and the strain levels. the purpose of this study was to apply selected dna fingerprinting methods to the identification and strain discrimination of t. rubrum clinical isolates. fifty - seven isolates from as many tinea patients were subjected to species identification by polymerase chain reaction restriction fragment length polymorphism (pcr - rflp) analysis and strain differentiation using a randomly amplified polymorphic dna (rapd) method, with two primers designated 1 and 6. using pcr - rflp, 55 of the isolates studied were confirmed to be t. rubrum. among those, a total of 40 and five distinct profiles were obtained by rapd with primers 1 and 6, respectively. the combination of profiles from both rapd assays resulted in 47 genotypes and an overall genotypic diversity rate of 85.4%. a dendrogram analysis performed on the profiles generated by rapd with primer 1 showed most of the isolates (87.3%) to be genetically related. pcr - rflp serves as a rapid and reliable method for the identification of t. rubrum species, while the rapd analysis is rather a disadvantageous tool for t. rubrum strain typing.
pulmonary arterial hypertension (pah) is a progressive, debilitating disease characterized by elevated arterial blood pressure in the pulmonary circulation that left untreated, results in right ventricular failure and death. its prevalence has been estimated to be between 15 and 50 cases per million and even with modern treatment, the prognosis remains guarded.1 in fact, data from the us registry to evaluate early and long - term pah disease management (reveal) showed 1- and 5-year survival of 87% and 57%, respectively, therefore, new and more effective therapeutic options are necessary to treat this severe and unrelenting disease.2 pah is pathologically defined by a mean pulmonary arterial pressure 25 mmhg at rest and pulmonary vascular resistance of > 3 woods units, in the absence of either an elevated wedge pressure or cardiac output. it is distinguished as group 1 according to the updated clinical classification of pulmonary hypertension and comprises a group of conditions and diseases with similar pathological findings, hemodynamic characteristics and in some cases treatment options (table 1).3 for the purpose of this review we will focus our discussion on the clinical utility and evidence on macitentan in pah. the development of effective treatment for pah has necessitated a clearer understanding of the pathophysiology of the disease. while the complete mechanism is still incompletely understood, it is clear that the development and maintenance of pah is secondary to a dysregulation of vascular tone. due to poiseuille s law these changes are mediated by vascular smooth muscle, which is influenced by three vasoactive molecules released by endothelial cells : nitric oxide (no) and prostacyclins, which induce vasodilation, and endothelins, which induce vasoconstriction. in healthy subjects, these mediators are in a dynamic balance to preserve an optimal pulmonary vascular tone.4 several experiments in both animal models and humans have demonstrated that pah is associated with reduced levels of prostacyclins and increased vasoreactivity to no.57 agents that target no metabolism via phosphodiesterase type 5 (pde-5) inhibition to increase cyclic guanosine monophosphate (cgmp) levels have shown great promise in long - term trials and are now an important part of pah therapy.8 there has been extensive interest in developing treatments that target no release or prostacyclin receptor activation but these efforts have run into difficulties with drug delivery and duration of action.4 there is also emerging technology of prostacyclin synthase gene therapy and cell - based therapy using native stem cells and engineered stem cells with enhanced prostacyclin production capacity and direct activation of the cgmp cascade.9 endothelin represents another well - known target in the treatment of pah. endothelins are 21 amino acid peptides, with three distinct isoforms and two known endothelin receptors (eta and etb) ; endothelin-1 (et-1) has been found in increased levels in the plasma and pulmonary vascular endothelium of patients with pah and has been implicated in the pathogenesis and progression of pulmonary vasoconstriction and eventual right ventricular failure in these patients.1012 endothelin receptor antagonist (eras) including ambrisentan (letairis), bosentan (tracleer), and sitaxsentan (thelin) have been designed and tested in patients with pah in randomized controlled clinical trials and have been shown to improve functional capacity, exercise capacity and delay disease progression in these patients.1315 of these, ambrisentan and sitaxsentan are eta - selective eras, while bosentan has nonselective activity on endothelin receptors. macitentan (opsumit) is a novel orally active dual era, which was recently approved in both the european union and us to delay disease progression and reduce hospitalizations in patients with pah. et-1 is expressed constitutively by endothelial cells and secreted from the basal surface of the vascular endothelium, where it promotes both local vasoconstriction and cell proliferation of the underlying smooth muscle as well as fibroblast proliferation ; changing tissue structure and inducing fibrosis.16,17 secretion of et-1 can be further promoted by hypoxia, shear stress, thrombin activity, or inhibited by the effect of no.18,19 it is first manufactured in the lung endothelial cells as an inert precursor, which is then activated by et - converting enzyme. it is then released in close proximity to the endothelial smooth muscle where it binds to endothelin receptors ; plasma levels of et-1 do not reflect the true paracrine activity of et-1 on these cells.20,21 et-1 promotes vasoconstriction and vascular remodeling by activating g - protein coupled endothelin receptors. the most important of the endothelin receptors are etar and etbr.4 the former is found on smooth muscle cells, fibroblasts, and cardiac myocytes, while the latter is expressed on both smooth muscle and endothelial cells.22 binding of et-1 to etar causes g - protein activation and increased intracellular inositol triphosphate levels, which causes calcium release and vasoconstriction in muscle cells.23 in contrast, et-1 binding to etbr causes release of no and prostacyclin, and inhibition of apoptosis for several cell lines, including vascular smooth muscle.24,25 despite their apparent contrasts, there appears to be significant roles for both etar and etbr in et-1-mediated pulmonary arterial vasoconstriction. in rat models, the combination of etar and etbr blockade resulted in maximal reduction in vasoconstriction by et-1.26 classically, endothelin receptors have been considered monomers that, when activated by et-1, signal the release of intracellular calcium via g - coupled protein and downstream cellular signaling. recent experimental observations have suggested synergy between etar and etbr, however, leading to the interesting proposal of significant cellular interaction between these two receptors. it is possible that the downstream activity of etar and etbr requires heterodimerization to activate the coupled g - protein, and that eras block this heterodimerization and prevent downstream activation by et-1, or that under conditions of selective etar antagonism, etbr is able to assume partial functions of its counterpart receptor.27 macitentan is a second - generation potent dual era with tissue targeting properties, its chemical name is n-5-(4-bromophenyl)-6-(2-[5-bromopyrimidin-2-yl]oxyethoxy)-n-(propylsulfamoyl)pyrimidin-4-amine - n--propylsulfamide (figure 1). macitentan displays high affinity and sustained occupancy of the etrs in human pulmonary arterial smooth muscle cells, with a 50-fold increased selectivity for the etar when compared to the etbr subtype. macitentan also demonstrates slower dissociation kinetics from endothelin receptors than other eras, which may underlie its observed differences in adverse effects from other eras.28 macitentan shares a similar side effect profile to other eras, but it possesses some important differences as well by virtue of its dissociation kinetics and chemical structure. it belongs to the sulfamide class, with a sulfur dioxide linked to two organic chains. it is metabolized by oxidative depropylation to act-132577 and by oxidative cleavage to act-373898 ; both of these reactions are catalyzed primarily by the cytochrome p450 (cyp3a4) system.29 of these, only the former is metabolically active, and at steady state contributes about 40% to macitentan s total potency.30 as its metabolism is driven by the cyp3a4 system, macitentan s serum levels can rise significantly with strong inhibitors of the p450 system like ritonavir or ketoconazole, or fall precipitously with strong inducers like rifampin.31,32 following the initial pharmacokinetics studies, it has been estimated that about 74% of macitentan s ingested dose is bioavailable, this bioavailability is constant regardless of food intake, age, sex, or ethnicity.31,32 greater than 99% of macitentan and its metabolites remains avidly bound to plasma proteins, and it is principally cleared by a combination of hepatic and renal pathways (50%), while fecal elimination accounts for another 25% of excretion. in terms of drug - drug interaction, a randomized, open - label crossover study by sidharta found that coadministration of macitentan and warfarin did not change either the international normalized ratio levels or factor vii activity compared to controls. also, the investigators found no significant changes in the pharmacokinetics of macitentan and sildenafil when administered together in healthy volunteers. neither severe renal impairment nor hepatic impairment (child pugh classes a, b, or c) were found to confer any additional risk with the drug.32 these were concluded after administering a single 10 mg dose of macitentan to sequential groups of eight subjects with mild, moderate, and severe liver disease and comparing the results to a control group without liver disease, although plasma levels were lower in patients with underlying liver disease, no significant difference in pharmacokinetics was found. similar evaluation was done in patients with severe renal impairment, defined as creatinine clearance 3 uln) in the treatment arm compared to placebo (3.6% in the macitentan 3 mg group, 3.4% in the macitentan 10 mg group, and 4.5% in the placebo group). when transaminitis was present, though, it tended to be more severe in the patients treated with macitentan ; transaminases > 8 uln were five - fold greater in the treatment arm (2.1%) compared to placebo (0.4%).42 therefore, it is currently recommended to monitor liver enzymes at the initiation of treatment and then in the presence of clinical findings, and to discontinue macitentan therapy if patients develop sustained aminotransferase elevations, bilirubin elevations > 2 uln, or severe liver injury. anemia has been a commonly reported side effect in clinical trials of all of the eras. the anemia is generally mild, rarely required treatment discontinuation, and usually stabilizes after about 12 weeks of therapy.37 the etiology of this anemia is unclear, but it does not appear to be related to hemolysis or hemorrhage. rather, it may be related to drug inhibition of an etbr - mediated protection against red blood cell apoptosis,46 or more likely due to hemodilution as a result of increased fluid retention.47 a dose - dependent increased incidence of anemia was seen in the seraphin trial (8.8% in the 3 mg group, 13.2% in the 10 mg group, and 3.2% in the placebo group). this anemia was severe enough to require drug discontinuation in two subjects, and resolved with cessation of therapy. similar to liver enzymes, it is recommended to measure hemoglobin concentration before starting treatment with macitentan and as clinically indicated thereafter. finally, macitentan can cause a number of minor neurologic, cardiac, and respiratory disturbances. the most common neurologic complaint reported is headache (4.6% more when compared to placebo), while in terms of cardiorespiratory side effects there was an increased incidence of upper respiratory tract infections and nasopharyngitis in patients on macitentan (4%). in addition, there was a dose - dependent increase in the incidence of bronchitis with macitentan (8% at the 3 mg dose, 11.6% at the 10 mg dose, and 5.6% in the placebo group).42 the initial studies with bosentan demonstrated an improvement in functional and hemodynamics parameter in subjects with pah but also showed significant hepatotoxicity (14% of patients had levels > 3 uln).1315 similar studies examined etar - specific antagonists like sitaxsentan and ambrisentan and found therapeutic benefit but once more at the expense of severe, and (in the case of sitaxsentan) life - threatening adverse effects.46,48 macitentan s effectiveness was established in the pivotal seraphin study in 2013.42 in this event - driven double - blind control study, investigators randomized in a 1:1:1 fashion 742 patients with proven diagnosis of pah to either macitentan 3 mg / day, macitentan 10 mg / day, or placebo. background pah therapy was also allowed, including oral and inhaled prostanoid therapy, calcium channel blockers, or phosphodiesterase-5 inhibitors. subjects included patients with idiopathic or heritable pah (56.8%), pulmonary hypertension due to shunts (8.4%), hiv (1.4%), connective - tissue disease (30.5%), or drug or toxin exposure (3%). hemodynamics and functional status were verified by right heart catheterization in all subjects (mean pulmonary artery pressure of 53.917.5 mmhg, mean pulmonary vascular resistance 12.8 woods units) and by 6 minutes walk test (mean 350100.2 meters ; world health organization [who ] class ii iv disease). the composite primary endpoint was time from initiation of therapy to the occurrence of the first pah - related event (worsening of pah, initiation of intravenous or subcutaneous prostanoids, atrial septostomy or lung transplantation) or all - cause mortality until the end of treatment. prespecified secondary end points included the change from baseline to month 6 in the 6-minute walk distance, the percentage of patients with an improvement in who functional class at month 6, death due to pah or hospitalization for pah up to the end of treatment, and death from any cause up to the end of treatment and up to the end of the study. following a median treatment duration of 115 weeks, 31.4% (n=76) patients taking macitentan 10 mg / day and 38% taking macitentan 3 mg / day (n=95) experienced a primary endpoint event, compared to 46.4% (n=116) of patients taking placebo (p 15% reduction in 6-minute walk distance, worsening of symptoms, and need for additional pah treatment with a relative risk reduction of 45% for the primary composite endpoint in patients taking macitentan 10 mg / day and 30% in patients on 3 mg / day when compared to placebo (hazard ratio [hr ] 0.55 [97.5% confidence interval { ci }, 0.39 to 0.76 ; p<0.001 ] and 0.70 [97.5% ci 0.52 to 0.96 ; p=0.01 by the log - rank test ], respectively). the number needed to treat to avoid one primary endpoint at 2 years was six patients. although benefit was shown in the secondary composite endpoint of death and hospitalizations due to pah, this benefit was mainly driven by pah - related hospitalization with a hr of 0.50 versus placebo in subjects taking macitentan 10 mg / day (97% ci, 0.34 to 0.75 ; p<0.001) and 0.67 versus placebo in subjects randomized to macitentan 3 mg / day (97% ci, 0.46 to 0.97 ; p<0.001). when isolated pah - related mortality data was analyzed, there was a trend favoring the intervention arm versus placebo, however, it was not statistically significant (hr 0.44 [97% ci, 0.16 to 1.25 ] 10 mg / day and hr 0.87 [97% ci, 0.37 to 2.04 ] 3 mg / day p=0.07) (table 2). at 6 months of treatment, a subset of 145 subjects underwent hemodynamic assessment, which demonstrated reductions in pulmonary vascular resistance and improvements in cardiac index in the macitentan group but not in the placebo group. the who functional class of patients in each group was also examined ; while all patients improved after 6 months of study from their baseline, there was a significant improvement in functional class in patients on macitentan compared to placebo (13% in placebo vs 20% at 3 mg [p=0.04 ] and 22% at 10 mg [p=0.006 ]). finally, in assessments of the 6-minute walk distance, there was a mean decline in the placebo group of 9.4 m while the treatment groups both showed improvements in 6-minute walk distance (+ 7.4 m in the 3 mg arm and + 12.5 m in the 10 mg arm). all of these benefits seemed to extend both to treatment - nave and previously treated patients.42 furthermore, a post hoc analysis by channick showed that macitentan 10 mg / day was associated with a reduced risk of both ; all - cause and pah - related hospitalizations and no increase in the risk for hospitalizations for other causes. in 2013, the us food and drug administration approved macitentan (opsumit) 10 mg once - daily for the treatment of pah in order to delay disease progression and reduce hospitalizations. the eu commission approved it for similar usage as monotherapy or combination therapy for pah in adult patients with a who functional class of ii to iii. given the drug s known teratogenicity, it bears a boxed warning as category x, and the possibility of pregnancy should be excluded in any female patient who will be taking the drug. while peripheral edema, hepatotoxicity, and anemia appear to be class effects of the eras, clinical trials of macitentan indicate that its unique pharmacokinetics decrease the incidence of these side effects significantly compared to previous eras. the 2013 seraphin trial also illustrated macitentan s efficacy in reducing the severity of pah in both treatment - nave patients and those on therapy, primarily in terms of disease progression and pah - related hospitalizations. in the us, it can be used in patients with hepatic and renal impairment without adjustment, though it has known teratogenicity and should be absolutely avoided in patients who may be pregnant. an open - label extension of the seraphin trial to examine the long - term effects of macitentan in patients with pah is currently underway, labeled seraphin - ol (nct00667823). there are also several trials underway to test macitentan s efficacy in treating other related diseases and conditions, for which its use is currently off - label. it is currently being studied in the phase ii merit (macitentan in the treatment of inoperable chronic thromboembolic pulmonary hypertension) trial (nct02060721) for its utility in chronic thromboembolic pulmonary hypertension. also, the phase ii melody-1 and -2 (macitentan in combined pre- and postcapillary pulmonary hypertension due to left ventricular dysfunction) trials are prospective, multicenter, randomized controlled studies to evaluate the safety and tolerability of macitentan in pulmonary hypertension due to left ventricular dysfunction. finally, macitentan is also being tested in congenital heart disease in the phase iii maestro (macitentan in eisenmenger syndrome to restore exercise capacity) trial, expected to conclude in march 2016. macitentan is also under investigation for any utility in other processes involving the endothelin axis, including as combination therapy for glioblastoma multiforme, as treatment for digital ulcers in systemic sclerosis, or as treatment for portopulmonary hypertension. pah remains to be a significant disease with long - term burden and clinical implications. macitentan is the newest era available for the treatment of pah, and has been shown in clinical trials to be well tolerated and clinically effective in both treatment - nave patients and those on background therapy. like other eras, it can be orally dosed once daily, and has a similar cost to other members of its class. unlike other eras, however, it does not appear to require dosage adjustments in patients with hepatic or renal impairment. although head to head studies and long - term follow - up data are needed to further establish its beneficial effects and tolerability, current evidence suggests that macitentan has a more favorable side effect profile than older eras and may therefore be a more attractive treatment option for patients with pah.	pulmonary arterial hypertension is a progressive, debilitating disease caused by a dysregulation of the pulmonary vascular tone that inevitably leads to right heart failure and death without treatment. until relatively recently, the treatment options for those afflicted by pulmonary arterial hypertension were limited ; today, a greater understanding of the pathophysiology behind this disease has led to several evidence - based therapies that can improve pulmonary function and quality of life for these patients. one of the primary mediators of pulmonary vascular tone is endothelin-1, which is a potent and long - lasting vasoconstrictor. macitentan is a second - generation endothelin receptor antagonist that acts selectively as a pulmonary vasodilator without the significant side effects noted with previous endothelin receptor antagonists. this review focuses on the mechanism of action and pharmacokinetics of macitentan, as well as the adverse effects, efficacy, and clinical uses of macitentan in the clinical trials to date. in addition, the authors briefly review clinical trials currently underway to illustrate possible future directions for the use of macitentan.
hepatitis c virus (hcv) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. hcv is primarily transmitted by blood - borne routes, including shared needles and transfused blood products. the who estimates that a minimum of 23% of the world s population is chronically infected with hcv (wasley and alter, 2000). despite the fact that hcv is targeted by innate, cellular, and humoral immune mechanisms, it establishes long - standing persistent infection in a majority of the people that it infects (pawlotsky, 2006). the virus forms small round - shaped particles ranging from 50 to 80 nm in diameter. the mature hcv virion is thought to consist of a nucleocapsid, an outer envelope composed of e1 and e2 viral proteins, and a lipid membrane. density gradient analyses have shown that viral rna exists within both low- and high - density fractions (andre., 2005), and that the low - density fractions contain lipoproteins that associate with apolipoprotein b (apob), apolipoprotein e (apoe), triglycerides, and cholesterol, as well as viral structural proteins (thomssen., 1992 ; andre., 2002 ; maillard., 2006 ; nielsen., only the low - density fraction derived from hcv - positive human serum exhibits high infectivity in chimpanzees (bradley., 1991 ; beach., 1992). since the establishment of a robust tissue culture infection system using strain hcv jfh-1 (lindenbach., 2005 ; wakita., 2005, the entire hcv lifecycle has been studied and the biophysical properties of the viral particles produced using the hcvcc cell culture system have been characterized. most viral rna - containing particles secreted from hcv - infected cells are poorly infectious and fractionate at high densities such as 1.14 g / ml, while highly infectious hcvcc are found within low - density fractions of 1.10 g / ml (lindenbach.,, we provide a general account of our current understanding of the hcv lifecycle and a review of recent studies focusing on the morphogenesis of hcv particles within cell culture systems. hepatitis c virus is a positive - stranded rna virus, and its 9.6-kb genome contains an open reading frame encoding a polyprotein of 3000 amino acids (aa) flanked by untranslated regions (utrs) at both ends. six genotypes have been reported based on hcv genome sequence variations (simmonds, 1996). the utrs are highly structured sequences encompassing critical cis - active rna elements essential for genome replication and translation. the 5 utr, which is 341 nucleotides (nt) in length, contains an internal ribosomal entry site, which is a prerequisite for cap - independent translation of viral rna, from which four highly structured domains (i iv) are produced (bukh., 1992 ; tsukiyama - kohara., 1992 ; wang., 1993 ; honda., 1996 ; friebe., 2001 the 3 utr varies between 200 and 235 nt in length, including a short variable region, as well as a poly(u / uc) tract with an average length of 80 nt which is considered crucial for rna replication, and a virtually invariant 98-nt x - tail region (tanaka., 1995 ; ito and lai, 1999 ; friebe and bartenschlager, 2002 ; yi and lemon, 2003). the genome is translated into a single precursor polyprotein, which is processed by cellular and viral proteases into 10 structural and non - structural proteins (core, e1, e2, p7, ns2, ns3, ns4a, ns4b, ns5a, and ns5b ; figure 1). hcv proteins derived from the amino - terminal third of the precursor include core, e1 and e2 structural proteins. a crucial function of core protein is assembly of the viral nucleocapsid. the aa sequence of this protein is well conserved among different hcv strains compared to other hcv proteins. the non - structural (ns) proteins ns3-ns5b are thought to assemble into a membrane - associated hcv rna replicase complex. ns4a serves as a cofactor for ns3 and is involved in targeting ns3 to the er membrane (wolk., 2000). ns4b plays a role in the remodeling of host - cell membranes, probably to generate a site for replicase assembly. ns5a is also thought to play an important but undefined role in viral rna replication. the role of hcv ns proteins in assembly of the infectious virion is described below. the hcv genome and polyprotein. post - translational cleavages by spp (signal peptide peptidase), sp (signal peptidase), ns2 - 3 pro (ns2ns3 cysteine protease), and ns3 pro/4a (ns3 serine protease and ns4a complex) lead to the production of functional hcv proteins. functions of each protein in the viral lifecycle are indicated below the open reading frame. the core is 191 aa in length and consists of three distinct predicted domains : an n - terminal domain that is two - thirds hydrophilic, a c - terminal domain that is one - third hydrophobic, and a short signal peptide sequence of the downstream protein e1. a c - terminal membrane - anchor of the core is further cleaved by a signal peptide peptidase. the mature core is estimated to be 177179 aa (ogino., 2004 ; okamoto., maturation of the core by a signal peptide peptidase is required for virion production (okamoto. biophysical techniques have been used to demonstrate that the mature core is a dimeric alpha - helical protein (boulant., 2005). its aa sequence is highly conserved among different hcv strains, compared with other hcv proteins. thus, core is used in most serologic assays since anti - core antibodies are highly prevalent among hcv - infected individuals. core protein is found on er membranes, on the surface of lipid droplets (lds), on the mitochondrial outer membrane, and to some extent, in the nucleus (moradpour., 1996 ; barba., 1997 ;, 1998 ; hope., 2002 ; suzuki., 2005). following is a proposed mechanism of translocation of core to membranes within the er network such as lds (mclauchlan. after being processed by a signal peptide peptidase, a large part of the core remains within cytoplasmic leaflets of the er membrane due to preservation of the original transmembrane domain. the cytoplasmic leaflets then swell as lipid accumulates between the two membrane leaflets. as a result, the core is translocated along with part of the er membrane to the surface of a nascent ld before the droplet buds off the er. the proteasome - dependent degradation pathway then participates in post - translational modification of the core (suzuki., 2001, 2009 ; ubiquitin ligase e6ap and proteasome activator pa28gamma are key regulators in determining the fate of the core, and thereby play a role in virus production (shirakura. the 120 n - terminal residues of the core protein (domain i) contain multiple positively charged residues that are implicated in rna binding and homo - oligomerization. it is therefore likely that this domain is a prerequisite for assembly of the hcv nucleocapsid (kunkel., 2001 ; it has been shown that a region extending from aa 72 to 91 is responsible for auto - oligomerization of core (nakai., 2006). although conclusive data for direct packaging of the hcv genome into the viral capsid is lacking, the viral rna sequence of the core protein through to the ns2 region appears not to contain a cis - acting packaging signal. a subgenomic replicon rna carrying the ns3ns5b region can be encapsidated in the viral trans - packaging system (ishii., 2008 ; steinmann., 2008 ; adair., 2009 ; masaki., in addition, the rna sequence encoding the first 62 aa of core contains highly conserved structures including two stem - loops that are important for rna translation / replication (mcmullan., 2007). domain ii (aa 120170) is predicted to form two alpha - helices that enable core to associate with membrane proteins and lipids. it has been proposed that domain ii folding occurs in a membrane environment and is critical for the folding of domain i (boulant., 2005). in addition, a cysteine residue at aa 128 of domain ii creates a disulfide bond to produce a core protein dimer that is required for particle formation (kushima., 2010). domain iii, pertaining to 20 residues at the c - terminal, is highly hydrophobic and serves as a signal sequence for e1. although little is known about the molecular mechanisms governing assembly of core into nucleocapsids, comprehensive mutagenesis studies have enabled identification of various aa residues which are essential for hcv morphogenesis (murray. two n - glycosylated envelope proteins e1 and e2 are exposed on the surface of the virus as a heterodimer that mediates viral attachment to host - cell receptors and facilitates virus entry (op de beeck., 2004 ; vieyres., 2010). ectodomains of e1 and e2 are translocated inside the er lumen and their transmembrane domains are inserted in the membrane of this compartment. e1 contains four to five n - linked glycans and e2 has 11 n - glycosylation sites. hcv glycans, which are thought to contain a mixture of complex and high mannose side - chain, play a role in envelope protein folding and formation of the e1/e2 complexes. the importance of incorporating n - linked glycans of the envelope proteins into infectious virions has been demonstrated (helle., 2010). a recent study has shown that hcv infection activates the er - associated degradation pathway, which in turn controls the fate of e1 and e2 and modulates virus production (saeed., 2011). a role of viral ns proteins in hcv production was first suggested following the observation that jfh-1-derived ns proteins are required to generate infectious virus from intra- and inter - genotype chimeric constructs (lindenbach., 2005 ; pietschmann., evidence supporting a role of ns2, ns3, and ns5a in the assembly or release of infectious hcv has come from mutational analyses (jones., 2007 ; miyanari., 2007 ; appel., 2008 ; jirasko., 2008 ; ns5a is a hydrophilic phosphoprotein which plays a key role in viral rna replication and is involved in modulation of cell signaling pathways and the interferon response (huang., 2007). ns5a is associated with membrane mediated by a unique amphipathic alpha helix which is located at its n - terminus (moradpour., 2005), and part of ns5a localizes to lds when expressed alone or as the viral polyprotein (shi., 2002). experiments based on hcv genomes containing mutated ns5a have shown that some mutants result in failure of association of ns5a with lds and failure of production of infectious particles (miyanari., 2007). further studies have revealed that the c - terminal region of ns5a plays a key role in hcv production (appel., 2008 ; masaki., 2008 ; tellinghuisen., substitutions at a serine cluster of the ns5a c - terminus (aa 2428, 2430, and 2433) which have no impact on viral rna replication, have been observed to inhibit the interaction between ns5a and core, thereby suggesting that an association between ns5a and core might be involved in virus production (masaki., 2008). structural analyses have shown that the n - terminal region of ns5a forms a claw - like dimer, which might accommodate rna and interact with viral and cellular proteins and membranes (huang., 2005 ; tellinghuisen., it appears that recruitment of ns5a to lds, thereby enabling interaction with the core, is crucial for virion assembly. one can imagine that newly synthesized hcv rna bound to ns5a is released from the replication complex - containing membrane compartment and can then be captured by core through interaction with the c - terminal region of ns5a at the surface of lds or ld - associated membranes. consequently, viral rna becomes encapsidated and virion assembly proceeds (figure 2). in this regard, a study has revealed an interaction between ns5a and apoe, suggesting that recruitment of apoe by ns5a is important for the assembly and release of hcv particles (benga., 2010). after accumulation of synthesized genome rna and the viral proteins, the hcv particles are assembled in an er - related compartment in close connection with the vldl pathway. the viral replication complex, which is composed of ns3ns5b and host factors, is a specialized structure protected by cellular membrane. newly synthesized viral rnas are recruited to surfaces of lipid droplet (ld), where er membrane is associated, possibly via interaction between ns5a and core. thus, the rnas enable to associate with core, thereby proceeding encapsidation and nucleocapsid formation. the nucleocapsid is presumably inserted into the lipid core of the luminal ld and buds into the er lumen with incorporation of e1 and e2. ns3ns4a is another component of the viral replication complex that exhibits serine protease, as well as rna helicase and rna - stimulated ntpase activity, necessary for viral rna replication. it is now apparent that ns3ns4a also contributes to viral assembly (yi., 2007 ; ma., 2008 ; han., 2009 ; a previous study has provided genetic evidence that two major subdomains of the ns3 helicase, one demonstrating ntpase activity and the other associated with rna binding, are involved in the early stages of virion assembly, independent of their roles as enzymes (ma. a separate investigation has revealed a contribution of the acidic domain of ns4a to both rna replication and virus assembly (phan., 2011). ns2 is a cysteine protease composed of a highly hydrophobic n - terminal membrane binding domain which forms either three or four transmembrane helices that insert into the er membrane. ns2 also contains a c - terminal globular and cytosolic protease subdomain, that produce zinc - stimulated ns2/3 autoprotease activity together with the n - terminal one - third of ns3. mutagenesis of ns2 has identified regions or residues that are important for infectious virus production (jones. for example, mutations involving the dimer interface of the protease region or the c - terminus of ns2 impair the production of infectious hcv, while the catalytic activity of ns2 is not required for viral assembly. genetic and biochemical data demonstrate that ns2 is possibly involved in multiple interactions with both hcv structural and ns proteins including e1e2, p7, ns3, and ns5a, suggesting that ns2 has a role in recruiting these viral proteins to sites in close proximity with lds. alternatively, it may act as a scaffold promoting virus assembly (jirasko., 2010 ; ma., 2011 ; p7 is a 63-aa polypeptide located between e2 and ns2, and is a membrane - spanning protein localized within the er. although p7 is not required for viral rna replication in cell culture, the protein is essential for hcv infectivity in chimpanzees (sakai., 2003). subsequent analyses in hcvcc systems have demonstrated that p7 is important for virion production since the introduction of p7 mutations, such as mutations in the basic residues required for its ion channel activity, impair virus production (jones. although it is not yet clear whether the ion channel function of the protein is needed for virus assembly, a recent study has demonstrated that p7 functions as an h channel in native intracellular membranes and links p7-induced ph changes to the production of infectious intracellular virions (wozniak., 2010). hcvcc contained within low - density fractions from the culture supernatant of virus - producing cells displays greater specific infectivity than virus in high - density fractions (lindenbach. looking also at the behavior of hcv circulating within the sera of infected hosts, it may be that low - density virus associates with lipid and very - low - density lipoprotein (vldl) and/or low - density lipoprotein (ldl). furthermore, hcv particles obtained from virus - infected animals, such as chimpanzees and chimeric mice transplanted with human hepatocytes, demonstrate greater infectivity than virus produced in cell cultures. the virus populations derived from these infected animals have been observed to fractionate into lower density fractions than a major population of hcvcc (lindenbach., 2006). interestingly, the association of cholesterol and sphingolipid with hcv virions has been shown to play a critical role in viral infectivity (kapadia. depletion of cholesterol and sphingomyelin from hcv virions inhibits the infectivity of hcvcc (aizaki., 2008). the structural requirement of virion - associated cholesterol for infectivity, as well as its influence on buoyant density, and the association of apolipoprotein with hcv, has been further demonstrated by yamamoto. (there is accumulating evidence that assembly and secretion of hcv particles are associated with the vldl assembly pathway. lipoproteins can be differentiated on the basis of their density, which is affected by lipid content and the types of apolipoprotein they contain. vldls are large triglyceride - rich lipoproteins (3080 nm in diameter) containing cholesterol, cholesteryl esters, apob, and other minor apolipoprotein(s). vldls carry triglyceride from the liver to peripheral tissue for storage and to provide a source of energy. triglyceride availability and the size of intracellular triglyceride pools are important regulatory factors in the regulation of vldl production. in addition, the microsomal triglyceride transfer protein (mtp), which is responsible for the transport of triglyceride and cholesteryl esters across er membranes, is required for vldl assembly. purified membrane vesicles containing the hcv replication complex are enriched with apob, mtp, and apoe (huang., 2007). moreover, agents that inhibit vldl assembly also inhibit hcv secretion from cells producing infectious virus (chang., 2007 ; apob, apoc1, and apoe have been observed to associate with hcv particles during viral morphogenesis in the hcvcc system (chang., 2007 ; meunier., 2008 ; jiang and luo, 2009 ; owen., 2009 ; benga.,, 2007 ; jiang and luo, 2009 ; owen., 2009 ; benga., 2010 ; hishiki., these findings demonstrate that apoe is important for hcv infectivity, suggesting that hcv virions are assembled as apoe - enriched lipoprotein particles. a study in which virion - associated cholesterol was depleted and replenished with exogenous sterol analogs has provided evidence that virion - associated cholesterol contributes to the interaction between hcv and apoe (yamamoto., 2011). apoe is a polymorphic protein with three major isoforms : apoe2, apoe3, and apoe4. differential roles of apoe isoforms on infectious hcv production have been revealed : ectopic expression of apoe3 or apoe4 enables recovery of infectious hcv, while apoe2 has little influence on virus production (hishiki., 2010). not surprisingly, apoe2 demonstrates significantly less ldl receptor binding activity than apoe3 and apoe4 (davignon., 1988). in fact, secretion of e1 and e2 within the culture supernatant is reduced by treatment with mtp inhibitors (icard., 2009). vldl maturation occurs by acquisition of lipids from lds either in the er lumen or in the golgi apparatus. it is likely that hcv envelopment takes place in a lipid - enriched microdomain at the er membrane where luminal lds or vldl precursors are generated (figure 2), in keeping with evidence of increased cholesterol content among hcv particles compared to host - cell membranes (aizaki., 2008). neutral lipids such as triglycerides and cholesterol esters are stored within cytosolic ld in cells. ld is thought to be a source of neutral lipid for metabolism and membrane synthesis. neutral lipids form the ld core and are surrounded by an outer layer of amphipathic lipids such as phospholipids and cholesterol. prior to the availability of a tissue culture system for virion production, hcv core was shown to associate with er membranes and on the surface of lds in heterologous expression systems in mammalian cells (moradpour., 1996 ; barba., 1997). early studies of cells infected with hcv jfh-1 indicate that core is detectable at the er and on the surface of lds in association with the er (rouille., 2006). core associates with lds in a time - dependent manner and disruption of this process coincides with a loss of virion production (boulant., 2007). it has subsequently been demonstrated that lds are directly involved in the production of infectious hcv and that core recruits viral non - structural proteins as well as the replication complex to the ld - associated membranes, suggesting that the association between core and lds is required at a certain stage of hcv morphogenesis (miyanari., 2007). fluorescent labeling and functional imaging of core in living cells has recently been used to visualize core during hcv assembly (counihan., 2011). core has been observed to move to the surface of large, immobile lds soon after protein translation. diacylglycerol acyltransferases (dgats) catalyze the final step of triglyceride synthesis and are crucial for ld biogenesis. a study has revealed that dgat1 interacts with core and is required for trafficking of the core to ld. disrupting translocation of the core to ld by inhibiting dgat1 activity or through knockdown of the dgat1 gene impairs virus production (herker., 2010). thus, there is now increasing evidence that lds play a central role in the production of infectious hcv and participate in virus assembly. however, one study has demonstrated that, while core derived from hcv jfh-1 is strongly associated with cytoplasmic lds, minimal core from a hcv clone with higher assembly efficiency is detectable on lds (shavinskaya., 2007). thus, it remains debatable whether hcv assembly is initiated on the surface of lds or at sites where er cisternae are in contact with lds. based on the current evidence, a model for nucleocapsid formation following the initial phase of assembly is demonstrated in figure 2. two potential models to explain hcv nucleocapsid formation, including the model shown in figure 2, have been proposed by bartenschlager. evidence regarding the assembly of infectious hcv particles has accumulated over the past several years since the availability of hcvcc systems. a variety of key factors in hcv morphogenesis have been identified, including the requirement for components of the vldl biosynthetic machinery and viral ns proteins. however, there are still essential questions to be answered. detailed mechanisms pertaining to nucleotide formation, genome packaging, and the way in which hcv interacts with the lipoprotein / vldl pathway, as well as the precise role of various ns proteins and p7 in hcv assembly, remain unclear. structural studies will be important to clarify the exact composition of the hcv virion, as well as similarities and differences between hcvcc generated in huh-7-derived cells and the lipoviroparticles produced by circulating virus within infected individuals. 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.	more than 170 million individuals are currently infected with hepatitis c virus (hcv) worldwide and are at continuous risk of developing chronic liver disease. since a cell culture system enabling relatively efficient propagation of hcv has become available, an increasing number of viral and host factors involved in hcv particle formation have been identified. association of the viral core, which forms the capsid with lipid droplets appears to be prerequisite for early hcv morphogenesis. maturation and release of hcv particles is tightly linked to very - low - density lipoprotein biogenesis. although expression of core as well as e1 and e2 envelope proteins produces virus - like particles in heterologous expression systems, there is increasing evidence that non - structural viral proteins and p7 are also required for the production of infectious particles, suggesting that hcv genome replication and virion assembly are closely linked. advances in our understanding of the various molecular mechanisms by which infectious hcv particles are formed are summarized.
precise, flexible, and specialized regulation of membrane trafficking is key to tissue patterning and differentiation during the development of multicellular organisms. it underlies the development of cell and tissue polarity (apodaca., 2012 ; farr., 2009 ; winter., 2012) and allows cells to specialize in the secretion or absorption of specific cargo in response to different signals or functional demands (cao., 2012 ;, it is essential to be able to visualize and manipulate specific membrane trafficking pathways. the rab protein family provides a unique entry point to study membrane trafficking pathways and their function in development and differentiation. rabs comprise a large family of lipid - modified gtpases that localize to specific subcellular membrane compartments. they cycle through gtp- and gdp - bound states, acting as molecular switches to recruit effector proteins that control compartment biogenesis (seabra and wasmeier, 2004 ; zeigerer., 2012), functional properties (liu and storrie, 2012) and composition (behnia and munro, 2005 ; zerial and mcbride, 2001), and direct vesicle motility (horgan and mccaffrey, 2011), tethering (sinka., 2008), and fusion (schimmller., 1998). a set of five rabs (1, 5, 6, 7, 11), which we denote the core rabs, has been maintained in almost all eukaryotes from unicellular organisms to metazoans, fungi, and plants (pereira - leal, 2008 ; pereira - leal and seabra, 2001). functional studies in yeast and in tissue culture cells have revealed their key functions in regulating the core secretory and endocytic pathways common to all cells. interestingly, recent genomic phylogeny studies have suggested that the putative last eukaryotic common ancestor (leca) had a much larger repertoire between 1523 rab proteins. this group includes the core rabs, but also many others that are lost in different eukaryotic lineages. in contrast, the rab family underwent a tremendous expansion correlated with the emergence of metazoans (bock., 2011 ; elias., 2012 ; klpper., 2012 ; pereira - leal and seabra, 2001). this expansion has been proposed to reflect the greater complexity of membrane trafficking pathways required for cell communication, tissue patterning, and differentiated cellular functions. systematic analysis of the tissue specificity and subcellular localization of rabs is an important first step in understanding how membrane trafficking pathways are organized in different cell types and how they are deployed during development and differentiation. they maintain distinct protein and lipid compositions on their apical and basolateral surfaces through targeted delivery, endocytosis, and recycling of specific cargo (apodaca., 2012). neurons also polarize trafficking of membrane and secreted proteins to organize the somatodendritic and axonal domains ; these have been suggested to rely on sorting mechanisms similar to the basolateral and apical domains of epithelial cells, respectively (dotti and simons, 1990 ; siegrist and doe, 2007). if so, are the rabs that first appeared in metazoans more likely to be deployed in this way ? much has been learned about the subcellular localization of rab compartments by expressing tagged rab proteins. however, unphysiological expression levels can alter membrane trafficking and distort the appearance of the relevant membrane compartments (mottola., 2010). we therefore generated a toolkit for the systematic analysis of rab protein expression and function in drosophila melanogaster. the drosophila genome predicts 33 rab proteins based on sequence similarity, and there is evidence that 27 are expressed (chan., 2011 ; zhang., 2007). fourteen of these rabs were already present in the leca (between 23 billion years ago), and the other 13 first arose by duplication and divergence at the root of the metazoan lineage (500 million years ago). while the genes encoding many rab proteins have undergone further expansions in vertebrates, this is not the case in drosophila (diekmann., 2011 ; elias., 2012 ; klpper., each drosophila rab exists in only a single copy, facilitating analysis (http://flybase.org)., 2008 ; rong and golic, 2000) to fuse yfp to the n terminus of each drosophila rab. furthermore, the yfp tag provides a target for knockdown approaches, allowing specific and controllable reduction of rab protein function (brankatschk., 2014 ; we systematically analyze rab protein tissue distribution and subcellular localization in six different tissues comprising over 23 cell types. we construct an online image database flytrab in which each rab can be viewed in whole tissues at subcellular resolution and a searchable annotation database for the systematic analysis and comparison of rab expression and localization (tomancak., 2007). analysis of these data provides insights into the evolution of membrane trafficking pathways and how they are deployed during cell polarization and tissue differentiation. to generate a novel set of endogenous n - terminally tagged rab proteins, we used homologous recombination to target the coding sequence for each drosophila rab gene and replace the predicted start codon with dna encoding yfp (figure 1a). western blotting indicates that the yfp tag does not alter rab4 protein levels (figure s1a). furthermore, tagging endogenous rab1, rab6, and rab7 with yfp does not perturb golgi morphology in salivary gland or fat body cells (figures s1b s1i). finally, except for yrabx5, all new yrab alleles produce viable homozygous flies, further confirming that their functions are not disturbed (figure 1b). to confirm the presence of yrab fusion proteins, and to examine their endogenous levels and proportions in different tissues, we performed semiquantitative western blotting against the yfp tag in three larval tissues : the fat body (fb), wing disc (wd), and salivary gland (sg) (figures 2a and 2b). we blotted equal amounts of tissue lysate from each homozygous yrab line and compared the signal strength of each yrab to a recombinant yfp standard (figures 2a and 2b). several yrabs (3, 14, 32, 26, x4, x5) were not detected in any of the three tissues (figures 2a and 2b). to confirm that these fusion proteins were produced at all, we blotted extracts of adult heads. this identified yrab3, yrab32, yrab26, yrabx4, and yrabx5, but failed to detect yrab14 (figures s2a and s2a). qualitatively, detectability of the different yrab proteins in wd, sg, and fb is consistent with mrna expression data from modencode (figure s2b). yrab14, yrab32, and yrabx4 are exceptions we can not detect these proteins in wd, fb, or sg although modencode (chen., 2014) predicts moderate expression. despite general qualitative agreement, yrab protein levels are not always proportional to reported mrna levels in modencode (figure 2b) it has been proposed that core rabs that have been conserved since the leca regulate pathways that are required in all cells, whereas rabs that first arose in metazoans regulate tissue - specific pathways. consistent with this, core rabs (yrab1, yrab5, yrab6, yrab7, yrab11) are present in all three tissues examined by western blotting (figures 2a and 2b). leca - rabs that are more often lost and those that arose in metazoans are more tissue - specific (diekmann., 2011)many of these are undetectable in some or all of the three tissues (figures 2a and 2b). we estimate that the most abundant yrabs (figure 2b, red) are present at concentrations of at least 10 m (for comparison, citrate synthase is present at 10 m) (srere, 1967), while the least abundant detectable yrabs (figure 2b, dark blue) are present at 20-fold lower concentrations. while rab gefs and gaps can modulate rab protein activity and flux through the corresponding compartment (cabrera and ungermann, 2013), rab protein concentration may also help determine the capacity of the compartment. if so, then a large fraction of secreted and transmembrane proteins move through pathways controlled by core rab proteins. finally, comparing yrab protein levels reveals unexpected heterogeneity in the proportions of even the highly conserved leca - rabs in different tissues. conserved rabs of the protein secretion machinery (rab1, rab2, rab6, rab8) are present in different proportions in wd, sg, and fb cells. similarly, rabs with conserved functions in endocytic trafficking and recycling (rab4, rab5, rab7, rab11) are also present in different proportions in different cell types. it will be interesting to see whether these differences are reflected by changes in trafficking through the corresponding compartments. to examine expression and subcellular localization in a broader range of cell types, we imaged larval wd, sg, fb, and brains, as well as adult ovaries and testes (janning, 1997). we collected an array of 3d tiles from each tissue and stitched them (preibisch., 2009) to form an image of the entire tissue at subcellular resolution. to analyze these data, we developed a hierarchical annotation scheme using a controlled vocabulary describing yrab expression and subcellular localization at the tissue and cell type level. the terms expressed/not expressed describe the presence of a yrab in a tissue or cell type. to describe differential yrab expression in particular cell types within a tissue, we use the annotation term elevated levels. we use three independent terms to describe subcellular localization in general : cortical, intracellular punctate, and intracellular diffuse. a diffuse appearance could reflect either non - membrane - associated yrabs or small vesicles below the resolution of the light microscope. a particular yrab might exist in different pools within the cell and therefore be described by one or more of these terms. for polarized cell types, we add polarity annotation terms that separately describe the polarized distribution of each of the three possible pools (cortical, punctate, diffuse). for example, a particular yrab might have a diffuse intracellular pool that is not polarized, but a cortical pool that is polarized. thus, each yrab in each tissue or cell type is characterized by multiple features. we annotated at total of 23 cell types in six tissues : three cell types in the cns, ten in ovaries of different stages, four in testes, three in sg, one in the fb, and two cell types in both early and late third instar wd. to perform hierarchical clustering of yrabs according to specific chosen features, we constructed a binary matrix that displays all features of every yrab / tissue combination. all the yrab imaging data and annotations, including the binary matrix can be accessed through the functional yfp - tagged rab (flytrab) database. (2007) and provides a link to collaborative annotation toolkit for massive amounts of image data (catmaid) (saalfeld., 2009) to browse the original 3d data set for each yrab and tissue type. the open source software catmaid (http://www.catmaid.org), originally used for em data, allows online browsing of terabyte - scale image data. we extended the capabilities of the catmaid software to allow visualization of multichannel immunofluorescence data. we also added tools that facilitate co - localization analysis, as well as cropping and downloading image data. only the five core yrabs are expressed in all three tissues examined by western blotting. to ask whether this principle held over a broader range of cell types, we surveyed the flytrab database. to help visualize these findings, we clustered yrabs according to their expression in different cell types (figures 2c and s2c). the y axis positions of the branch lines in the dendrogram indicate the similarity of cell - type expression between the two groups connected by the line. yrabs connected by a branch line with a similarity level of 1 are expressed in identical cell types. the set of yrabs that are expressed in all annotated cell types is highlighted in red. yrabs that separate at lower levels of similarity (orange and yellow branch lines) are increasingly dissimilar to the ubiquitous group (i.e., they are expressed in fewer cell types). yrabx5 and yrab32 form an out - group since they are not expressed in any annotated cell type (figure 2c, gray). yrab 5 is not detectable in migrating border cells, but is present in all other cell types. in contrast, leca yrabs that are often lost during evolution (figure 2b, group iv) tend to be expressed in fewer cell types, as do yrabs that arose in metazoans. the metazoan yrab26, yrab27, yrab3, and yrabx4 form an extremely tissue - specific group (figure 2c, yellow). together, these yrabs are expressed in only four annotated cell types and share the common feature of being expressed in neurons. yrab26 and yrab27 are expressed identically only in neurons, whereas yrab3 and yrabx4 are expressed in one and two additional cell types, respectively. these data generally support the idea that core rabs organize the basic membrane trafficking machinery common to all cells, whereas other rabs are more likely to have tissue - specific functions. to investigate the relationship between rab protein localization and cell polarity, we examined the subcellular distribution of each drosophila yrab in the neurons of the early second instar cns and in three epithelial cell types with different functions (early third instar sg cells, ovarian follicle cells of different stages, along with early and late third instar wd cells). different cell types within the cns can be easily identified by a combination of brain morphology and immunostaining. the cns is surrounded by a layer of specialized glial cells that constitute a blood brain barrier (bbb). neuronal cell bodies are located peripherally and extend axons and dendrites into more central regions, forming a neuropil. cell bodies of primary neurons, which have already formed synaptic connections, are located closest to the neuropil. secondary neurons are beginning to be born, and their cell bodies lie in more outer regions. nuclei of both primary and secondary neurons stain with the post - mitotic marker elav. pluripotent neuroblasts and ganglion mother cells are mostly located in very peripheral regions, are large, and do not stain for elav. thus, we can easily compare the relative proportions of different yrabs in neuroblasts, cell bodies of primary and secondary neurons, and neuronal projections (figure s3a). nine of the 22 yrabs detectable in neurons of the larval cns are roughly equally distributed between cell bodies and the neuropil (figures 3i3q and s3j, black). six yrabs (1, 7, x1, 4, 6, 39) are enriched in neuronal cell bodies (figures 3c3h and s3j, blue). seven yrabs (3, 19, 23, 26, 27, 30, x4) are clearly enriched in the neuropil (figures 3r3x and s3j, magenta). compared with yrabs that are uniformly distributed or enriched in cell bodies, yrabs that are enriched in the neuropil comprise predominantly metazoan yrabs. this group includes the highly cns - specific yrab3, yrab26, yrab27, and yrabx4. yrab3 and yrab27 have well characterized roles in synaptic vesicle release (mahoney., 2006 ; sdhof, 2013), and it would be interesting to examine whether other neuropil - enriched rabs may have related functions. mrna localization to growth cones and to synaptic termini is thought to contribute to axon guidance and synaptic plasticity. these mrnas appear to be locally translated, indicating that dendrites and axons have a functional er / golgi protein synthesis machinery (jung. do these outposts rely on the same rab machinery as er / golgi assemblies in the cell body ? examining yrab protein distribution shows that canonical golgi and er rabs (rab1, rab2, rab6) are present in both cell bodies and the neuropil (figures 3 and s3j). thus, canonical rab machinery likely regulates transport of secreted and membrane proteins in neuronal projections. does membrane trafficking change as cells differentiate ? to examine this, we looked for yrabs with different expression levels in neuroblasts, neurons, and bbb glia. the expression of most yrabs (e.g., yrab6) does not differ between these cell types (figures s3c and s3c). however, neuroblasts express higher levels of some yrabs (4, 18, 21, 40) compared to neurons and bbb glia (figures s3d s3f and flytrab). these include not only tissue - specific yrabs (x4, 3, 26, 27) but also yrab5 (figures s3g s3i and flytrab). in contrast, yrab9 is undetectable in neurons and neuroblasts, but is expressed in bbb glia (figures 3b and 3b). thus, the differentiation of glia and neurons may entail changes in membrane trafficking resulting from altered rab protein expression. to analyze rab protein localization in an epithelium specialized for apical secretion the post - mitotic sg cells produce and release digestive enzymes and, later, glue proteins into the apical lumen. each sg cell contains multiple large cup - shaped er / golgi compartments, which stain for the golgi marker lava lamp. these occupy the medial and basal regions of each cell, but are excluded from the most apical regions (szul., 2011). to help visualize the different yrab localization patterns in the sg we clustered them according to annotation features describing the polarized distribution of cortical, punctate, and diffuse pools (figure 4 g). as expected, yrabs with known functions in er - golgi trafficking (yrab1, yrab2, yrab6) localize to large compartments in the basal / medial cytoplasm (figures 4d4e, s4n, s4o, and s4 t). several yrabs (19, 30, 11) are enriched in a diffuse apical pool (figures 4b4c and 4 g, magenta, and s4i s4k). yrab11 does not cluster identically with yrab19 and yrab30 because it is also present in larger medial and basal structures like yrab39 and yrab7 (figures 4 g, green, and s4k s4m). the only yrab that is basally polarized in the sg is yrab4 (figures 4f, 4f, 4 g, blue, s4s, and s4s). the apical group of rabs (yrab11, yrab19, yrab30) are interesting candidates for mediating apical secretion in sg cells. interestingly, yrab19 and yrab30 are also enriched in neuronal projections, which may indicate a common function in polarized secretion in these cells. rab30 localizes to the golgi in s2 cells in contrast to its apical localization in sg cells. its interaction partners in s2 cells include not only golgins, but also components of the exocyst a complex that targets golgi - derived vesicles to the plasma membrane (gillingham., 2014). if rab30 mediates delivery of vesicle the golgi to the plasma membrane, then its different localization in the sg and s2 cells may simply reflect steady - state differences in its distribution. to what extent is rab compartment architecture a conserved feature of polarized epithelia ? to address this, we examined other epithelia, beginning with the follicle epithelium (fe) of the ovary. when egg chambers emerge from the germarium, the fe is cuboidal and its apical surface contacts the germline cells. fe cells proliferate between stages 2 and 6, and their growth and patterning is controlled by apical signals from the enlarging germ cells (gonzlez - reyes and st johnston, 1998 ; lpez - schier and st johnston, 2001). at stage 6, fe cells stop dividing and begin to grow by becoming polyploid. at stage 8, fe cells contacting the oocyte start to elongate, becoming columnar over increasingly larger regions as the oocyte enlarges. at the same time, fe cells overlying the nurse cells flatten and become squamous. to visualize intracellular yrab protein distribution features in early (stages 28) and late (stage 9) fe, we clustered them according to annotations describing the polarized distribution of cortical, punctate, and diffuse pools (figures 5i and 5j). yrab proteins in the fe display two different architectures depending on the developmental stage : during early stages, the compartments organized by yrab4, yrab5, yrab7, yrab11, yrab19, yrab30, yrab39 and yrabx1 polarize apically toward the overlying germline (figures 5b5c and 5i, magenta). however, as the fe becomes columnar by stage 9, a new rab architecture develops. apical yrab polarization is lost (figures 5f and 5f), and yrab1, yrab2, yrab6, yrab7, yrab8, yrab10, and yrab18 now polarize toward the basal side of the cell (figures 5g5h and 5j, blue). basal yrabs fall into two distinct groups in the dendrogram (figure 5j, blue) because their compartment morphologies differ. comparing dendrograms depicting subcellular localization of yrabs in the sg, early fe, and late fe reveals no consistent pattern (figure s5). although the early fe and sg share the feature that yrab11, yrab19, and yrab30 localize apically, this feature disappears in the late fe. furthermore, yrab4 is basal in the sg and apical in the early fe. to analyze rab compartment organization in a fourth epithelium, we turned to the larval wd, which later gives rise to the adult wing and thorax. the wd is a folded epithelial sac with an apical lumen and a basal side that is bathed in hemolymph. as the disc grows, it becomes pseudostratified on one side and squamous on the other a feature common to many other developing epithelia such as the neural tube and retina. we examined and annotated yrab localization in third instar wd before and after pseudostratification (figure 6 and flytrab). before pseudostratification yrab5 and yrabx1 are apically enriched, and yrab8 is basally enriched (flytrab and figure 6f). yrab1, yrab2, yrab4, yrab5, yrab7, yrab8, and yrabx1 become apically localized (figures 6c, 6c, and 6 g, magenta), while yrab11 (figures 6d, 6d, and 6 g, magenta / blue) localizes to the extreme apical and basal ends of the cells. more than half of the yrabs in the wd are reproducibly represented in a characteristic structure found in the apical cytoplasm at the level of the junctions (figures 6d6f and 6 g, dashed line). it contains not only apically enriched rabs like yrab1 (figures 6c, 6c, and 6 g, magenta dashed line), but also rabs with an otherwise uniform distribution such as yrab6 (figures 6e, 6e, and 6 g, black dashed line). strikingly, many rabs in both the secretory (rab1, rab2, rab6, rab8) and endocytic pathways (rab4, rab5, rab7, rab11) are represented in this apical hub even when they also localize to other positions in the cell. their localization is consistent with independent markers of er and golgi compartments (figures 6h6i). the dramatic rab redeployment during pseudostratification is clearly seen by comparing the dendrograms shown in figures 6f and 6 g. taken together, systematic analysis of rab localization in these epithelia reveals little similarity and provides no support for the idea that apical - basal epithelial polarization relies on a common rab compartment architecture. to illustrate this dissimilarity, we clustered yrabs according to features describing polarity and compartment morphology, generating a dendrogram for each tissue (figure s5). these clustering patterns highlight the differences between epithelial tissues and even between different developmental stages of the same tissue. thus, epithelial cells deploy different subsets of rab compartments in a polarized fashion depending on their specialized tasks. to examine whether rab tissue distribution and subcellular localization was related to rab protein phylogeny, we performed clustering analysis based on annotation terms that describe compartment morphology and polarity in different cell types (figure s6). this revealed several groups of rabs with strong similarities in localization and cell type expression. comparing this analysis with sequence - based phylogenetic trees shows that two of these groups (rab19 and rab30 and rab3, rab26, rab27, rabx4) consist of rabs that are evolutionarily related, but that diverged in the earliest metazoans. the fact that compartment morphology / polarity and tissue distribution of rabs within these groups have evolved together for the last 500 million years suggests that their functions are interdependent. other rab pairs with similar subcellular and cell type distribution are not related phylogenetically (e.g., rab23 and rab35). their shared features, however, suggest that it would be interesting to investigate whether they have similar functions. yrab23 and yrab35 represent a phylogenetically unrelated rab pair with similar subcellular localization patterns. these rabs are both found at the cell cortex in all cell types where they are present (figure s6, yellow box). we therefore wondered whether they might fulfill redundant functions in tissues where they are co - expressed. while yrab35 is generally expressed, yrab23 is mainly expressed in epithelial tissues such as the developing wing (flytrab). rab23 mutants disturb tissue polarity in the adult wing (pataki., 2010). normally, each wing cell produces a single, distally oriented hair. when rab23 is lost, cells produce multiple hairs and hair orientation shifts away from the proximal - distal axis toward the wing margin (figure 7b). however, dominant - negative rab35 expression and targeted rab35 knockdown disturb actin organization in thoracic bristles (zhang., 2009). anti - gfp rnai and anti - gfp nanobody expression have been used to knock down endogenously gfp- and yfp - tagged proteins (brankatschk., 2014 ; caussinus., 2012 ; neumller., 2012). to ask whether these methods were generally effective at reducing levels of yfp - rabs indeed, both methods strongly reduce levels of yrab23 (figures s7a s7c) and produce phenotypes similar to rab23 mutants (figures 7e, s7j, and s7k). although gfp rnai efficiently reduces yrab35 levels (figure s7e), its depletion causes only occasional subtle disturbances in hair morphology (figure 7d and data not shown). to look for interactions between rab23 and rab35, we knocked down both rabs in the developing wing blade of yrab35;yrab23 homozygous flies using nubbingal4. g) produces defects in hair polarity and morphology that would be expected from the sum of the individual knockdowns. the additional removal of yrab35 does not enhance hair polarity defects caused by loss of yrab23. however, knockdown of both rabs produces a phenotype not seen in either knockdown alone : the formation of ectopic cross veins (figures 7f and s7h). thus, rab23 and rab35 function redundantly to organize the pattern of veins in the wing. membrane trafficking is key to the regulation of many signaling systems that determine the wing vein pattern (restrepo., 2014 ; wang and struhl, 2004). it will be interesting to further investigate how rab35 and rab23 influence signal transduction. here, we describe a genetic resource for visualization of endogenous rab proteins. we provide a catmaid - based annotated 3d image database (flytrab) that allows the user to browse rab protein distribution with subcellular resolution in 6 different tissues comprising over 23 cell types. dedicated search algorithms connect image data with the defined vocabulary in annotation trees and allow clustering of rabs according to features of interest. all original recorded data are accessible and downloadable online a feature that will allow users to investigate specific aspects of interest more deeply. furthermore, hypotheses generated by searching this database can be easily tested with knockdown strategies that target the yfp tags. these will provide powerful tools to explore the different functions and specializations of membrane trafficking pathways in vivo. we exploited the comprehensive nature of this resource to address questions in cell differentiation, cell polarity, and rab protein evolution. our analysis shows that cell differentiation is accompanied by quantitative and qualitative changes in rab protein expression and localization, suggesting that rewiring of membrane trafficking pathways is a key feature of differentiation. for example, quantitative western blotting of fbs, sgs, and wds revealed not only tissue - specific rab protein expression, but also changes in the relative levels of rabs that control basic cellular processes such as secretion, endocytosis, and recycling. furthermore, neuronal differentiation is accompanied not only by expression of neuron - specific rabs involved in synaptic vesicle release (rab3, rab26, rab27), but also quantitative changes in levels of other rab proteins. 2014) and it would be interesting to know how changing their relative proportions influences the endocytic pathway in neurons. rab4, rab18, and rab40 are more highly expressed in neuroblasts than in neurons. interestingly, rab18 mutations have been associated with warburg micro syndrome, which is characterized by postnatal microcephaly (bem., 2011 ; cheng., 2014. it would be interesting to know whether neural stem cells function normally in patients with this syndrome. bbb glial cells also derive from neuroblasts (desalvo., 2011). rab9 expression increases with differentiation of bbb glia, suggesting that it may have special functions there. finally, not only do rab levels change as tissues develop their subcellular localization can change as well. rab proteins undergo a striking redeployment during development of both the ovarian follicular epithelium and the wd epithelium. interestingly, although rab proteins throughout the wing pouch re - localize as cells become pseudostratified, there appear to be few obvious differences in their expression levels or distribution within the wing pouch despite the fact that this region is already being patterned by the activity of several different morphogens (crozatier. the single exception is rabx6, which is expressed at elevated levels in the anterior compartment and in sensory organ precursor cells (flytrab). thus, morphogen spreading and signaling probably operates in a field of cells with relatively homogeneous membrane trafficking capabilities. although we initially wondered whether epithelial polarity might depend on a standard polarized membrane trafficking architecture (farr., 2009 ; weisz and rodriguez - boulan, 2009), our observations do not support this idea. while many different rabs can assume a polarized localization in specific epithelia at specific developmental stages thus, different polarized distributions of rab compartments may simply reflect differences in epithelial function. for example, one set of rabs (4, 5, 7, 11, 19, 30, 39, x1) is apically localized in early follicle cells in the ovary, but not at later stages. apical localization of these rabs correlates with a time of intense communication between the germline and the overlying follicle cells that is mediated by notch / delta signaling a pathway whose regulation critically depends on endocytosis and recycling (deng., 2001 ; this may account for the enrichment of endocytic / recycling rabs (4, 5, 7, 11) in the apical regions of these cells. the apical polarization of rabx1, rab39, rab19, and rab30 suggests that they could also be involved in communication with germline cells. in later egg chambers follicle cells overlying the oocyte undergo columnarization, during which the area of the basolateral membrane increases. by this time, the same set of rabs is no longer apically localized. instead, a large number of rabs in the secretory pathway (1, 2, 6, 8, 10) as well as three others (7, 11, 35) become basally localized. it would be interesting to investigate whether increased basal delivery of secretory vesicles expands the basolateral membrane in these cells. completely different changes in rab protein architecture and polarity occur during pseudostratification of the wd. during pseudostratification, many rabs (1, 2, 4, 5, 7, x1) become apically localized including rab8, which reverses its polarity. pseudostratification also correlates with the development of a novel cluster of rab compartments located at the level of apical junctions, which we call the apical hub. this structure coincides with an apical web of microtubules (eaton., 1996) that arises during pseudostratification in response to dpp signaling (gibson and perrimon, 2005 ; shen and dahmann, 2005). although this area of the cell is only a few microns in diameter, over half of the rabs expressed in the wd are reproducibly represented here. thus, the apical cytoplasm of wing epithelial cells is a major membrane trafficking hub. it will be interesting to discover whether the apical hub is a general feature of pseudostratified epithelia and whether it might facilitate rapid intercellular communication during patterning of these developing tissues. finally, these studies show that the ability to polarize is not limited to rabs that arose first in metazoans ; almost all leca and metazoan rabs are distributed in a polarized way at least in some cell types. the only widely expressed rabs for which we never detected a polarized distribution were rab21 (present in the leca but often lost) and rab9 and rab40 (that evolved in metazoans). this suggests that the metazoan expansion of the rab family provided a wider variety of membrane trafficking routes that could be targeted in different ways, rather than specific pathways required for cell polarity. they generally rapidly diverge in their expression patterns, acquire mutations, and may eventually adopt novel beneficial functions (neo - functionalization). duplicates may also divide functions once present in a single ancestral protein (taylor and raes, 2004 ; zhang, 2003). our analysis has revealed two instances in which a group of phylogenetically related rab proteins show extremely similar patterns of cell - type - specific expression and even subcellular localization. one group comprises rab19 and rab30 and the other rab3, rab26, rab27, and rabx4. the members of both groups diverged from each other over 500 million years ago (elias., 2012). this suggests that there has been selection to preserve their coordinate expression and subcellular localization, and their functions somehow depend on each other. when a rab is duplicated, both rabs are recruited to the same compartment by the same signals. of course the rab specifying the derivative branch might not function without the rab that specified the original branch, explaining the requirement for coordinate expression. in the future, the tools for rab visualization and knockdown described here will facilitate these and other analyses. yfp - tagged rab alleles were generated by ends - in homologous recombination and the initial genomic duplication was resolved using the i - cre system as described by rong and golic (2000) and maggert. embryo collections and staging protocols were performed at 25c ; flies were raised on conventional cornmeal agar under a 12 hr light/12 hr dark cycle at 25c. nubbin - gal4 (# 42699) and uas - gfprnai (# 41559 and # 9331) are available from bloomington stock center, rab from dgrc (# 125902). uas - gfpnanobody flies were provided by the affolter lab and en(105)-gal4 flies are from c. dahmann. larval brains (4448 hr after egg collecting [aec ]), salivary glands (7280 hr aec), early (6872 hr aec), and late (110120 hr aec) wing discs were dissected in ice - cold pbs (cns, salivary glands) or graces medium (wing disc), fixed with 4% pfa at room temperature and probed with one or more of the following : anti - gfp (invitrogen), dapi (roche), anti - hrp - cy5 (dianova), anti - elav (developmental studies hybridoma bank [dshb ]), anti - dlg (dshb) or anti - crbs2.8 (gift from e. knust). the larval fat bodies (7280 hr aec) were fixed with 4% pfa for 30 min at room temperature and stained with anti - gfp (invitrogen), anti - dlg (dshb), and dapi (roche). anti - lva (gift from e. knust) and anti - kdel (enzo life science) were used to mark organelles. aged (57 days) flies were fed for 1 day at room temperature (rt) with yeast paste ; ovaries and testis were dissected and fixed with 4% pfa in pbs and stained for 3 days at room temperature with anti - gfp (invitrogen), phalloidin-555 (roche), anti - dlg (dshb), and dapi (roche). all samples were mounted in vectashield mounting medium (vector labs) and photographs were acquired with an olympus1000 confocal microscope and evaluated using fiji imaging software. catmaid image data sets were analyzed manually and annotated using a defined terminology to describe subcellular localization of individual yrabs (see supplemental experimental procedures). multiple annotation features that are represented in a binary matrix characterize the tissue distribution and subcellular localization of each yrab. we used this binary matrix to perform hierarchical clustering of yrabs according to specific annotation features using past 3.01 (http://folk.uio.no/ohammer/past/).. rooted trees (dendrograms) were generated according to similarity coefficients based on unweighted pair group method with arithmetic mean (upgma) linkage (see supplemental experimental procedures). wing discs, fat bodies, and salivary glands from feeding 3 instar larvae (110120 hr aec) were dissected in ice - cold graces medium, homogenized with a plastic pestle in 1% tx-100 pbs lyses buffer, and pelleted at 20,000 g for 5 min at 4c. protein content from recovered supernatants was measured using bca (manufacturer protocol, invitrogen) and 8 g protein lysatelane were loaded on 12.5% sds - pages. additional blots (figure s1) were probed for rab4 (gift from m. zerial) and -tubulin (gift from e. knust). comparing yrab signals to recombinant yfp standards shows that there is more than 10 ng of the most abundant yrabs per lane, corresponding to 0.2 pmol. each lane contains four wing discs, and a wing disc has a volume of 0.005 l.	summarymembrane trafficking is key to the cell biological mechanisms underlying development. rab gtpases control specific membrane compartments, from core secretory and endocytic machinery to less - well - understood compartments. we tagged all 27 drosophila rabs with yfpmyc at their endogenous chromosomal loci, determined their expression and subcellular localization in six tissues comprising 23 cell types, and provide this data in an annotated, searchable image database. we demonstrate the utility of these lines for controlled knockdown and show that similar subcellular localization can predict redundant functions. we exploit this comprehensive resource to ask whether a common rab compartment architecture underlies epithelial polarity. strikingly, no single arrangement of rabs characterizes the five epithelia we examine. rather, epithelia flexibly polarize rab distribution, producing membrane trafficking architectures that are tissue- and stage - specific. thus, the core machinery responsible for epithelial polarization is unlikely to rely on polarized positioning of specific rab compartments.
the picornavirus 5 untranslated region (5 utr) has proven the most suitable genomic target for molecular detection (8,9). at the time of this study, only 5 aiv complete genomes were available in genbank, complicating selection of reliable oligonucleotides for universal detection. a nested rt - pcr encompassing the aiv 5 utr was developed (table 1) and used for screening of stool samples collected in northern germany from outpatients with gastroenteritis. the first subcohort of this collection consisted of 499 patients with gastroenteritis ; samples were collected evenly from january through december 2004 in a prospective study on acute community - acquired diarrhea by 47 general practitioners in bremen, northern germany (10). the second subcohort consisted of 39 control patients without symptoms of gastroenteritis seen by the same physicians. the third subcohort consisted of 118 patients with diarrhea linked to outbreak scenarios involving canteen food (n = 36), kindergartens (n = 54), or retirement homes (n = 28). aiv, aichi virus ; i d, identification ; rt - pcr, reverse transcription pcr ; utr, untranslated region. 25-l qiagen onestep rt - pcr reactions as described by the manufacturer (qiagen, hilden, germany) used 400 nmol / l each of 1st - round primers, 1 g bovine serum albumin, and 5 l rna extract. amplification involved 30 min at 50c ; 15 min at 95c ; 10 cycles of 20 s at 94c, 30 s starting at 60c with a decrease of 1c per cycle, and 50 s at 72c ; and 40 cycles of 20 s at 95c, 30 s at 54c, and 50 s at 72c ; and a final elongation step of 5 min at 72c. 50-l platinum taq reactions as described by the manufacturer (invitrogen, karlsruhe, germany) used 1 l of 1st - round pcr product, 2.5 mmol / l mgcl2, and 400 nmol / l each of 2nd - round primers. amplification involved 3 min at 94c and 45 cycles of 20 s at 94c, 30 s at 60c, and 40 s at 72c. 25-l qiagen onestep rt - pcr reactions used 3 l of rna extract, 600 nmol / l of each primer, and 320 nmol / l of the probe. (darmstadt, german) 7700 sds instrument involved the following steps : 55c for 15 min, 95c for 15 min, and 45 cycles of 95c for 15 s and 58c for 30 s (fluorescence measured). we purified viral rna from stool samples by using the qiagen viral rna and dna stool mini kits (qiagen, hilden, germany) as described (8) ; 9 samples were positive for aiv by nested rt - pcr. the 5 utr sequences of these viruses were determined and deposited in genbank (accession nos. gq927704gq927712). with additionally available sequence data, real - time rt - pcr targeting conserved regions of the viral 5 utr was developed (table 1). assay sensitivity was determined to be 1.5 copies per reaction by using photometrically quantified in vitro crna transcripts, as described (9). retesting of all samples with this highly sensitive assay increased the aiv detection rate, yielding 10 positive samples. all case - patients were part of the subcohort of symptomatic outpatients seen by general practitioners (figure 1). in this cohort, no patients from the control group (n = 39) or from foodborne outbreaks (n = 118) had positive test results for aiv. however, this difference was not statistically significant for any group comparison (fisher exact test, 2-tailed p>0.05 for all). as shown in table 2, no other virus commonly associated with diarrhea was detected in any patient, including norovirus, rotavirus, adenovirus, astrovirus, parechovirus, or enterovirus. a bacterial cause of disease was ruled out by using standard culture methods (10). no clear association with a foodborne etiology, sociologic risk factor (including travel history), or contact with animals was observed. as shown in table 2, high rna copy numbers of up to 1.32 10 per gram of stool (mean 1.32 10, median = 1.82 10) were found in patients with positive test results. age distribution of cohorts tested in study of aichi virus in patients with acute diarrhea, germany. a) patients from food - associated diarrhea outbreaks (kindergartens, canteens, or retirement homes) ; b) outpatients seen for gastroenteritis by general practitioners ; c) nongastroenteritis control patients for the outpatient study cohort. arrows indicate patients who had positive test results for aichi virus by real - time reverse transcription pcr. no patients had fever (> 38.5c) or co - infections. all samples were tested for norovirus, rotavirus, adenovirus, astrovirus, parechovirus, enterovirus, and common bacterial pathogens. although samples had been collected throughout 2004, all aiv - positive cases occurred during 8 weeks from october to december and originated from a geographically restricted area within the city of bremen. to verify if this temporal and geographic accumulation of cases represented a point - source outbreak, we amplified and sequenced the entire viral protein (vp) 1 gene, which is commonly used for picornavirus typing, from 9/10 samples (genbank accession nos. failure of vp1 amplification in sample d / vi2591 was probably caused by low virus concentration. as shown in figure 2, a total of 8 samples formed a distinct phylogenetic cluster within aiv genotype b. the first 3 strains, sampled from october 19 through november 10 (table 2), were almost identical in vp1, with only 1 strain (d / vi2244) diverging by 2 synonymous substitutions. all other samples (november 16december 7) showed a vp1 nucleotide diversity of up to 0.8% (27/864 nt) and an amino acid diversity of up to 1.4% (14/288 residues) in comparison to the 3 initially sampled specimens and to each other. strain d / vi2287, sampled november 8, belonged to aiv genotype a, with nucleotide and amino acid differences of up to 13.2% and 5.4% from the genotype b strains. neighbor - joining phylogeny of aichi virus (aiv) viral protein 1 gene of strains from study of aiv in patients with acute diarrhea (boldface), germany, compared with strains from genbank. the tree was generated by using mega4 (www.megasoftware.net) using the maximum - composite likelihood nucleotide substitution model and complete deletion option. porcine kobuvirus was used as an outgroup (branch truncated as indicated by slashed lines). for many of the recently described picornaviruses, human pathogenicity is still under study. with the advent of metagenomics although it is generally difficult to generate sufficiently large and appropriately sampled control groups in studies on respiratory and enteric diseases, quantitative data on virus shedding and proof of monocausality can contribute to confirm the link of novel viruses to human disease. for aiv, the high concentrations found in several samples in this study provide support for viral replication in humans. however, virus shedding appeared unrelated to the severity of symptoms, and clinical presentations were generally mild. contrary to the findings of previous studies from france and tunisia (5,6), no food association could be observed in this study. in agreement with some, but not all, published reports (5,11), no case of aiv - associated gastroenteritis from this study had apparent co - infections, further supporting aiv pathogenicity in humans. the overall 2.0% detection rate of aiv in stool samples from outpatients with gastroenteritis is compatible with detection rates in recent studies from several european and asian countries (5,12). this was in sharp contrast to parechoviruses and cardioviruses detected predominantly in patients 75.0% described for cardioviruses and parechoviruses (14,15). the geographic and temporal accumulation of cases, together with the observed sequence variation, supports locally and temporally restricted circulation of aiv with human - to - human transmission, rather than a point - source epidemic pattern as observed in foodborne infections. oral human - to - human transmission would be facilitated by the high fecal virus concentrations in some patients. our data indicate that aiv can be considered an authentic human pathogen that can be transmitted from human to human.	we assessed aichi virus shedding in patients with gastroenteritis and negative test results for other viral and bacterial infections. high concentrations of up to 1.32 1012 rna copies / g stool were found in 10 (2.0%) of 499 outpatients sampled in northern germany, 2004. these data substantiate aichi virus pathogenicity in humans.
some 1,303,100 americans are in 18,900 nursing homes nationwide. over 86 percent of these residents are elderly (nchs, 1977). although this figure represents only 5 percent of the total population age 65 and over, some 20 percent of the aged will enter a nursing home before dying (lavor, 1979). in 1977, nursing home expenditures amounted to $ 12.8 billion, an estimate that excludes many medical services (such as most physician, services) provided to nursing home residents. (tables 1 and 2 present expenditure data through 1979. however, because the 1978 and 1979 data are preliminary, this presentation focuses on 1977.) government expenditures accounted for 57.2 percent of total nursing home outlays in 1977 ; private payment accounted for the remaining 45.6 percent. medicaid is the predominant public source of financing, accounting for 86 percent of the $ 7.3 billion in public expenditures. other public sources, such as the veterans administration and various state and local programs, compose the remaining 9 percent. the predominant source of private funding consists of direct out - of - pocket payments by nursing home residents and their families. third - party payments account for only 1.6 percent, and other private payments, such as charitable contributions, account for another 1.4 percent. the public - private split is essentially the same (54.6 percent public and 45.4 percent private). however, whereas private insurance pays for only 1.6 percent of private nursing home expenditures, it pays for 33.8 percent of private hospital expenditures. the private insurance sector has decided that nursing home services, except for some short - stay, acute patients, are not an insurable risk, at least at present. these estimates actually understate the dependence of the nursing home population on public sources of financing, for two reasons. first, because of various limitations on reimbursement, payment levels under medicaid and other public programs tend to be below the charge levels for private patients. as a result, the proportion of publicly supported patients is higher than the proportion of expenditures some 59.4 percent of residents are publicly supported at any one time. second, many medicaid beneficiaries in nursing homes become eligible via the spend - down provision ; that is, they enter the nursing home as private patients and subsequently become eligible only after reaching a poverty level by spending much of their income and divesting themselves of most of their assets. because medicaid beneficiaries in nursing homes tend to have long lengths of stay, and thus consume resources that are not covered by private insurance at a high rate, they are more likely than users of other medicaid services to have had middle class incomes prior to admission. hence, medicaid nursing home benefits are available to a broader segment of the population than other covered benefits. the growth in nursing home expenditures has been enormous. in three years out of the five - year period ending 1977, they have composed the fastest rising component of personal health care expenditures (table 3). between 1973 and 1977, expenditures grew 77.5 percent, compared with increases in the consumer price index (cpi) of 36.3 percent and in the gross national product of 44.4 percent. these increases place major pressures on the public sector to economize. at the same time, the growth in private out - of - pocket payments, combined with the unavailability of private insurance coverage, could create conflicting pressures on the public sector to do more. this expenditure growth reflects two factors increases in the cost per day and increases in the number of days used. data on per diem costs are available only for homes that are certified to participate in medicare or medicaid. between 1973 and 1977, these costs rose 55.2 percent, 50 percent faster than the cpi. the number of residents increased 21.1 percent, compared to a 12.7 percent growth in the elderly (over age 65) population. furthermore, the above data exclude medicaid expenditures for intermediate care facilities for the mentally retarded (icf - mr), which grew from $ 165 million in 1973 to $ 871 million in 1977. while recent expenditure increases are dramatic, future expansion could be even more intense because of the anticipated growth in the elderly population. currently, the social security rolls are netting an additional 600,000 people each year. whereas the total population is projected to grow by 40 percent between 1977 and 2030, the elderly population will more than double. nursing home use increases dramatically among those over the age of 75, and the proportion of the aged who are over 75 is rising. by 2035, that percentage is expected to increase from 38 percent to 45 percent (bureau of census, 1977). what are the implications for the demand for nursing home services ? table 4 displays the 1977 nursing home population by age cohort, starting at age 45. it also displays the percent of each age cohort that is in nursing homes. by applying the same percentage to the predicted populations by age cohort for the years 2000 and 2030 although age - specific use rates could change over time, it is instructive to analyze the impact of current use rates applied to predicted populations. these are displayed in table 5, which shows an increase in the number of nursing home residents of 54 percent by the year 2000 and 132 percent by the year 2030, assuming current age - specific use rates. essentially all of the publicly - financed portion of nursing home expenditures, and part of the privately financed portion, will be borne by the segment of the population that is of working age. some 88 percent of the functionally disabled between ages 18 and 64, and 70 percent of the elderly disabled, live with others. (callahan, 1980) it has been estimated that, for every person in a nursing home, there are as many as two persons who are equally disabled and who are living in other settings, mostly at home (shanas, 1971). the willingness of spouses and adult childern to care for their aged and disabled relatives has perhaps been underestimated in the popular press. nonetheless, the concern exists that this willingness and capacity may decrease as a consequence of increasing divorce rates, declining birth rates, the growth of single parent families, and women 's increasing participation in the labor force. indeed, some of the increases over the last decade in reported expenditures for long - term care including nursing home care may be an artifact of our system of national accounts. the gross national product (gnp) measures only the financial value of market transactions. it does not reflect the value of care given by family members, which may have decreased over the last ten years. it has been suggested that significant savings could be achieved by treating many of the patients who are now in nursing homes in the community. various studies estimate the number of residents who are candidates for treatment outside of the institution at between 10 and 20 percent in skilled nursing facilities and 20 and 45 percent in intermediate care facilities (congressional budget office, 1977). there are, however, reasons to be skeptical that such savings can be realized. first, there is evidence of a shortage of nursing home beds for public patients in some parts of the country. an urban institute study demonstrated that utilization was constrained in several states by the availability of beds (scanlon, 1980). while the study used data from 1969 and 1973, the situation is unlikely to have changed, since the growth in the number of nursing home beds has not kept pace with the growth in the elderly population. between 1973 and 1977, the number of nursing home beds increased 5.6 percent compared to a 12.7 percent growth in the population over age 65. hence, efforts to deinstitutionalize would merely result in other patients entering the nursing home. another reason for caution relates to the underlying basis for need for nursing home services. need is determined not by a medical diagnosis per se but by a combination of a functional impairment and some physical dependence on others. the availability of (usually unpaid) friends or family members is critical in the assessment of whether a person can live in the community. that is why nursing home use rates are nine times higher among unmarried than among married elderly persons (scanlon, 1979). national survey data for 1977 show that less than 10 percent of the nursing home population is not dependent on others for assistance in one or more of the following : bathing, dressing, using the toilet, mobility, continence, and eating (nchs, 1977). although a portion of the remaining 90 percent might be cared for in the community, the expense involved could be substantial. indeed, the complex problems in defining need may account in large measure for the limited private insurance coverage of nursing home and other long - term care services. finally, the cost experience borne out in a number of studies does not offer much hope that savings can be achieved, at least under current reimbursement mechanisms. each of these studies raises methodological issues, and they can be discussed only briefly in this paper. although additional research is needed before we can draw definitive conclusions, some broad patterns do emerge. one set of studies examines how public expenditures would be affected if individuals in nursing homes were to be cared for outside of the institution. these studies typically conclude that a significant proportion of patients can be treated at lower cost in the community. most such studies entail estimates of the cost of providing medical care outside the institution, based on a review of medical records. one study, of skilled nursing facility (snf) patients in minnesota, estimated both the fraction of snf residents who could be cared for at lower cost in the community and the cost savings that would accrue to the state (greenberg, 1974). specifically, the cost of housing, food, and so forth was included in calculating the cost of care in the different settings analyzed. in contrast, most studies have included these costs only for nursing home patients. also, the greenberg study recognized that the relative costs of home care versus institutional care vary with the level of impairment. greenberg concluded that 9 percent of minnesota 's present snf residents could be cared for at lower cost in the community and that minnesota could save approximately $ 400,000 annually by providing their care in the lower cost setting. the suggestion was made, but not tested, that similar analysis of an icf population, which is less impaired, might yield even greater savings. the second set of cost - effectiveness studies more closely approximates the effect on the use and associated expenditures of providing expanded coverage. unlike the previously - cited research, it focuses on the actual behavior of medicare and/or medicaid beneficiaries using alternatives to institutionalization, such as adult day care, homemaker services, chose services, and so forth. these studies analyze demonstration projects which were established to test the cost - effectiveness of expanded in - home and community - based services. some report a reduction in the use of institutions, whereas others report an increase. however, many of those that do report a reduction find that the cost of the community care significantly exceeds any savings in institutional expenditures. for example, william weissert examined several demonstration projects that provided homemaker and adult day care services to medicare beneficiaries (weissert, 1980). in each site analyzed, patients were randomly assigned to two groups : an experimental group that was covered for the new services in addition to existing medicare benefits and a control group that was not. the specific services provided to the experimental group varied slightly among projects but generally included social services, personal care, supportive services, and home management. weissert found that coverage of these services did not reduce the likelihood that recipients would use hospital or snfs. hospitalization was slightly higher for the experimental group, and snf utilization was the same for both groups. importantly, the total cost per beneficiary, including those costs associated with the new benefits, averaged $ 3,432 (or 60 percent) higher for the experimental group. unlike homemaker services, adult day care was available to beneficiaries who had not been hospitalized. the services were medically oriented but also included transportation, personal care, and a variety of social services. in contrast to homemaker services, these added benefits were accompanied by reduced use of both hospitals and snfs (10 versus 13 hospital days and 4 versus 9 snf days). however, the experimental group had an annual average net medicare cost of $ 6,501, compared to $ 3,809 for the control group, a net increase of 71 percent. several demonstration projects have been initiated since the completion of the experiments analyzed by weissert. many of these projects focus on the cost - effectiveness of expanding community - based and in - home services to medicaid - eligible populations. most of these projects are still ongoing, and the preliminary findings are not entirely consistent. the monroe county long - term care program in new york conducts comprehensive assessment of patient needs prior to admission to a long - term care facility (eggert and bowlyow, 1979). the project reports that the per diem costs of placing their clients at home has been generally 50 percent or less of the medicaid rate for comparable institutional care. however, we do not currently know whether the availability of broader benefits generated an increase in beneficiaries seeking services. the georgia alternatives health services program also uses patient screening and referral for medicaid eligibles but provides a wide array of new services, including adult day rehabilitation, social services, board and care, and adult foster care (georgia dma, 1979). finally, washington state 's community based care program found that broader coverage increased total costs 11 percent at one site and 4 percent at the second site, despite a decline in the medicaid nursing home population (solen, 1979). on balance, the projects are not confirming a substantial cost - savings from such interventions uniformly across sites. furthermore, studies that do report cost - savings often focus the cost analysis solely on medicaid costs. these studies fail to consider certain public expenditures that are usually higher outside of the institution, specifically various welfare and social security payments, housing support, and social services programs. in summary, nursing home expenditures will rise significantly. expanded programs of community services are highly desirable, but not as cost saving measures, since services would undoubtedly be used by persons not presently receiving formal or covered long - term care as well as by persons presently covered in institutions. while the availability of noninstitutional services might well improve the living conditions of impaired individuals, it should be treated as a probable addition to more than substitute for services currently covered by public programs. as pension plans improve and people become more aware of their own potential future long - term care needs, private sources of support could expand. however, the pressures for expanded publicly - financed support of nursing home and other long - term care services will remain intense. the response to these pressures is likely to be influenced by overall spending patterns on behalf of the aged. the president 's 1981 budget includes $ 158 billion to assist the aged in a variety of ways. much of the remaining 74 percent of the budget is for expenditures on behalf of all americans for example, defense and transportation rather than for any particular segment. the funds targeted for the elderly represent a 15 percent increase over the previous year, compared to an increase in overall outlays of 9 percent and a projected gnp growth of 10.71 percent. in addition, social security cash benefits, which are by law indexed for inflation, will increase 14.3 percent this year, and the number of beneficiaries will increase another 2 percent. the potential for ever increasing the share of the gnp that is transferred from the working population to the aged could be a major source of social tension over the next generation, particularly if the economy goes through substantial periods of stagnation. as a society, however, we must confront some increasingly difficult issues of how much money the working population should spend on behalf of the aged ; how that amount should be distributed among various functions, such as cash payments, acute medical care, long - term care, and social services ; and how the long - term care sector should be financed. rising nursing home expenditures are likely to contribute to making long - term care the most problematic area of social policy over the next generation.	nursing home care has become a major governmental responsibility. public expenditures for nursing home care amounted to $ 7.3 billion in 1977. they represented 57.2 percent of the $ 12.8 billion nursing home bill nationally and 12 percent of public spending on all personal health care. nursing home care absorbs more than one - third of all medicaid expenditures.this paper explores expenditure patterns in recent years and discusses some of the factors that will influence these patterns in the future. first we analyze recent trends over the five - year period ending 1977. then we project future utilization based on current age - specific use rates. finally, we review recent studies on the potential cost savings of noninstitutional alternatives to nursing home care.
the first us case associated with the epidemic in hispaniola was laboratory confirmed on november 15, 2010, in a us resident who had traveled to haiti and returned to florida. the first case in a patient with history of travel to dominican republic was laboratory confirmed on january 29, 2011. as of april 4, 2011, a total of 23 cholera cases associated with the hispaniola epidemic had been confirmed (figure 1). patients resided in florida (10), massachusetts (4), new york city (4), kansas (1), michigan (1), north carolina (1), virginia (1), and texas (1) (figure 2). illness onset dates ranged from october 23, 2010, to february 2, 2011. median age was 38 years (range 984 years), and 43% were female patients. confirmed cholera cases (n = 23), by onset date and travel history, united states, october 21, 2010february 4, 2011. geographic distribution of cholera cases in the united states associated with hispaniola, october 21, 2010april 4, 2011. all patients were treated with antimicrobial agents, rehydration, or both ; 9 (39%) were hospitalized, 6 (30%) sought care at an emergency department, and none died. six patients had illness onset before returning to the united states, 5 had illness onset on the day of return, and 12 had illness onset 111 days after return (typical incubation period for cholera is 18 hours5 days) (5). all 20 isolates matched the haiti isolate outbreak pattern by pulsed - field gel electrophoresis. susceptibility results for antimicrobial drug tested showed that all isolates were resistant to trimethoprim / sulfamethoxazole, furazolidone, nalidixic acid, sulfisoxazole, and streptomycin, and 18 isolates showed intermediate resistance to chloramphenicol, ampicillin, or amoxicillin / clavulanic acid. thirteen patients reported recent travel to haiti (median length of stay 7 days, range 254 days) and 9 to dominican republic (median length of stay 4 days, range 29 days). one patient reported no recent travel but consumed cooked conch brought to the united states from haiti by relatives. travel was reported to the following departments in haiti : artibonite (2), ouest (7), centre (1), nord (1), and sud (1). one case - patient traveled to 2 departments, and 2 did not specify a destination. all case - patients associated with the dominican republic had attended a wedding in la romana province on january 22, 2010 ; an investigation conducted by the dominican republic ministry of health is ongoing. aside from 2 case - patients who traveled to this wedding together, no other case - patients reported traveling together. visiting friends or relatives was the main reason for travel to haiti (table 1). four patients traveled to haiti to participate in relief activities, 2 as medical volunteers, 1 on a mission trip, and 1 to distribute canned foods. a wide range of exposures was reported (table 2) ; 5 patients were exposed to persons with cholera or cholera - like illness and to other risk factors for cholera acquisition. one volunteer reported no apparent lapses in safe water and food practices, although detailed information about food preparation was not available. water exposures include local lake and stream of water running down a street in haiti ; a pool in the dominican republic ; ocean (dominican republic) ; unspecified location in port - au - prince, haiti ; and a tank at a medical center in haiti. sources included newspaper articles (4), friends (4), cdc traveler s hotline (1), and the world health organization website (1) ; 2 patients reported > 1 source. two patients reported receiving a travel health alert notice upon arrival in the united states (m. selent, unpub. six months after the hispaniola cholera epidemic started in haiti, 23 associated cases were recognized in the united states. all cases were associated with recent travel to hispaniola or with consumption of seafood from haiti. the risk for cholera transmission in the united states is low because of improved water and sanitation, and there is no evidence of secondary transmission. florida, new york, and massachusetts have the highest populations of persons of haitian or dominican ancestry (6). most cases were reported from florida, the state with the largest haitian population. however, case - patients also resided in states with small haitian and dominican populations. travel between the united states and haiti is straightforward ; 4 us airports offer daily direct flights from florida and new york to port - au - prince. many persons, including many of haitian descent, traveled from the united states to haiti to help with the response to the january 2010 earthquake in port - au - prince. person - to - person transmission of cholera has only rarely been reported ; cases in medical workers are almost always attributable to consumption of contaminated food or water. person - to - person transmission is not clearly supported for either of the cases we report in medical workers, although it can not be ruled out. continued surveillance and detailed investigation of cases in medical workers is warranted to further define the risk, if any, of person - to - person transmission. echoing the latin american cholera epidemic in the 1990s, travelers to cholera - affected areas should be aware of the risk and should follow prevention measures to avoid infection. in particular, travelers visiting friends or relatives may be at higher risk for travel - associated infection (7). few case - patients had received cholera prevention education (educational materials available at www.cdc.gov/cholera/index.html) ; no cholera vaccine is licensed in the united states. until cholera in haiti and dominican republic resolves, clinicians, microbiologists, and public health workers in the united states should be prepared for more cases in travelers returning from hispaniola.	cholera is rare in the united states (annual average 6 cases). since epidemic cholera began in hispaniola in 2010, a total of 23 cholera cases caused by toxigenic vibrio cholerae o1 have been confirmed in the united states. twenty - two case - patients reported travel to hispaniola and 1 reported consumption of seafood from haiti.
morgagni foramen is a para - retrosternal defect resulting from an incomplete fusion of the septum transversum and sternum with the anterior ribs. surgical treatment consists of direct closure of the diaphragmatic defect, suturing by transabdominal or transthoracic access. we report a patient with hernia of morgagni who underwent a laparoscopic reduction and diaphragmatic defect closure. a formerly healthy, 69-year - old woman was seen at our department in february 1995 because of epigastric pain and subocclusive symptoms for nine months. a computed tomography showed a gross diaphragmatic anterior hernia with partial right and transverse colon migration (figure 1). the diagnosis of hernia of morgagni was made, and the patient was considered for repair of the diaphragmatic defect by the laparoscopic approach. the herniated bowel was gently pulled down with grasping forceps and placed entirely into the abdominal cavity (figures 2, 3 and 4). the defect was ovoid and was closed with interrupted polyester stitches (ethibond - ethicon) using the external knot - tying technique. trocars were retrieved under direct endoscopic vision, and the fascial incision was closed with glycolic acid (vicryl - ethicon). a chest xray showed the successful laparoscopic closure of the diaphragmatic defect (figure 6). laparoscopic view of hernia of the foramen of morgagni containing colon and omentum. colon and omentum chest x - ray done after operation, showing no evidence of morgagni 's hernia. after 12, 24, 36 and 48 months follow - up, the patient was symptom - free, without recurrence of her morgagni 's hernia (figure 7). chest x - ray done four years after the laparoscopic repair, showing no evidence of hernial recurrence. a formerly healthy, 69-year - old woman was seen at our department in february 1995 because of epigastric pain and subocclusive symptoms for nine months. a computed tomography showed a gross diaphragmatic anterior hernia with partial right and transverse colon migration (figure 1). the diagnosis of hernia of morgagni was made, and the patient was considered for repair of the diaphragmatic defect by the laparoscopic approach. the herniated bowel was gently pulled down with grasping forceps and placed entirely into the abdominal cavity (figures 2, 3 and 4). the defect was ovoid and was closed with interrupted polyester stitches (ethibond - ethicon) using the external knot - tying technique. trocars were retrieved under direct endoscopic vision, and the fascial incision was closed with glycolic acid (vicryl - ethicon). a chest xray showed the successful laparoscopic closure of the diaphragmatic defect (figure 6). chest x - ray done after operation, showing no evidence of morgagni 's hernia. after 12, 24, 36 and 48 months follow - up, the patient was symptom - free, without recurrence of her morgagni 's hernia (figure 7). chest x - ray done four years after the laparoscopic repair, showing no evidence of hernial recurrence. the defect is usually small and contains a sac with herniated omentum, transverse colon and, more rarely, liver, small bowel and stomach. acute symptoms are rare and are almost always due to large bowel obstruction. in infants, respiratory distress and cyanosis plain roentgenograms usually differentiate the hernia of foramen of morgagni from other masses (lung or mediastinal tumors, pericardial fat pad, pleural, pericardial, mediastinal or diaphragmatic cyst) or pathologies (atelectasis, pneumonia). barium enema, computed tomography and magnetic resonance may be required to confirm the diagnosis. although complications are rare, because of potential strangulation, hernia of morgagni foramen should be repaired. after viscera reduction into the abdominal cavity, the sac can or can not be excised. both the laparotomic and thoracotomic approach require a long postoperative recovery period, with significant mortality and a prolonged rehabilitation period. conversely, the laparoscopic approach for treatment of morgagni hernia results in an immediate return to normal diet and activities. literature review of laparoscopically treated morgagni 's hernia is reported in table 1. regarding the technique of defect closure, in a previous report kuster has underlined that the hernial sac does not needed to be removed. this removal, in fact, may result in massive pneumomediastinum with potential respiratory and circulatory complications. in kuster 's technique, the diaphragmatic defect was closed by a nonabsorbable monofilament with continuous suture joining the subcostal and retrosternal peritoneum to the full thickness of the diaphragmatic edge. then the suture was percutaneously brought back anteriorly to the abdomen. a 2-cm skin incision was made, through which the two ends of the suture were tied in subcutaneous tissue. more recently, fernandez - cebrian has described a patient in whom the hernial sac was removed without complications. also, in this case, the defect was repaired with a continuous suture, but with intra - abdominal knotting. in our own experience, the sac was not removed to avoid the unacceptable risk of damage to the pericardium and/or the mediastinic or diaphragmatic pleura. cases of fatal pneumopericardium have been reported after dissection of the peritoneal sac in children. we preferred to close the defect with interrupted nonabsorbable suture using an extra - abdominal knot - tying technique. in fact, in our own experience and in the literature, it has been noted that separate stitches are preferable to avoid tissue tearing. moreover, extracorporeal knotting has been shown to be easier to perform and is less time consuming than intracorporeal techniques. the drainage is generally left in place of an empty cavity, according to traditional principles of general surgery. probably, it is not useful, but, since we did not have any previous experience with this kind of operation, a prudential approach was preferred. rau in 1994, huntington in 1996, orita in 1997, and del castillo in 1998 reported a successfully repaired laparoscopic morgagni hernia by stapling a mesh prosthesis. rau did resect a peritoneal sac, and the prosthesis was covered with a flap of falciform ligament and with ligament teres. huntington did not resect the sac, and the prosthesis was covered by a peritoneal reflection obtained by a peripherical incision for several centimeters around the defect. in the orita experience also, the sac was not removed, and the operation was conducted by a gas - less approach to facilitate the suturing technique. vinard, in 1997, presented a case that allowed satisfactory surgical repair by simple closure of the hernial orifice with a running suture. because of the lack of experience reported in the literature, it is not possible to define whether or not mesh placement is better than a suture for closing the morgagni 's hernia. it is, however, noteworthy that the classic repair by the laparotomic approach is the direct suture of the linear hernial orifice. moreover, laparoscopic repair can be successfully associated with other procedures, such as cholecystectomy. follow - up of operated patients was reported only by some authors and only in one case for 24 months. it is not known whether or not patients were recurrence free after longer follow - up. in the patient herein reported, follow - up was done for more than four years and showed the absence of symptoms or recurrence. incidental ; preop = preoperative ; nr = not reported ; y = yes ; n = non ; rs = running suture ; ss = separate stitches ; a = stapled agraphes ; d = drainage. independently from the laparoscopic surgical technique used, literature data and our own experience indicate that the therapeutic and rehabilitative advantages that are well proved for cholecystectomy and other videolaparoscopic procedures with respect to a laparotomic approach can be extended to patients with hernia of foramen of morgagni as well.	the videolaparoscopic repair of a diaphragmatic hernia of morgagni by external knot tying technique is described. a 69-year - old woman with subocclusive symptoms by intrathoracic migration of abdominal viscera had an immediate and complete postoperative recovery. the hernial sac was not excised. a four - year follow - up shows no hernia recurrence. this case indicated that the laparoscopic approach can be considered a suitable and safe procedure for treatment of morgagni 's hernia.
concern over the rising cost of health care services in the united states has encouraged an extensive examination of every sector of the nation 's health care system. expenditures on drugs and drug sundries reached $ 22.4 billion in 1982 (gibson., 1983). although this is a sizeable amount, it is nonetheless small when compared with expenditures for hospital or physician services. however, these expenditures are seen as more amenable to control than some of the larger sectors, and several regulatory and competition - stimulating programs have been instituted at the federal and state levels of government to reduce drug costs. an important effort to contain the cost of prescription drugs has concentrated on encouraging the substitution of less expensive brands or generic drugs for more expensive brand name drugs. to allow a wider range of substitution to take place, most states have enacted some form of legislation modifying antisubstitution laws which now permit pharmacists to dispense drug products other than those prescribed. california is one of the states that has amended its antisubstitution law and has actively promoted drug substitution. the purposes of this article are to examine the extent of substitution, the resulting effect on the retail price of drugs, and the degree to which cost savings on less expensive brands or generics are passed on to consumers. in the first section, the origin of prescription drugs and state antisubstitution laws are briefly discussed. in the next section, the california substitution law requires pharmacists who dispense a different brand or generic drug rather than the brand name version prescribed, to pass on to consumers the resulting cost savings. to evaluate the compliance of pharmacists with the law, econometric models of drug retailing are developed and estimated in the third section of the paper. in the first 20 years after passage of the pure food and drug act in 1906, sales of medicinal drugs increased 600 percent. unlike the situation today, drug marketing was directed primarily at patients rather than the physicians ; less than 5 percent of drug advertising was directed at physicians, implying drug product selection was usually made by patients (and perhaps pharmacists) rather than by physicians. before the great depression, about 5 percent of drug sales was obtained directly from physicians and only one - quarter of drug sales from drugstores was prescribed by physicians (temin, 1979). all nonnarcotic drugs could be purchased without a prescription until 1938, when the federal food, drug, and cosmetic act was signed by president roosevelt. subsequently, two classes of nonnarcotic drugs prescription and over the counter were recognized. the distinction between the two was not precisely made in the 1938 act but was generally accepted. however, the legality of requiring prescriptions was unsettled until 1951 when the durham - humphrey amendment was passed. since then, physicians have assumed greater responsibility for choosing drug products. this shift has been noted by the pharmaceutical industry which, in 1972, spent $ 721.8 million promoting drug products (schwartzman, 1976). new categories of wonder drugs were introduced in the market during the 1940 's and 1950 's, and the pharmaceutical industry became increasingly concerned about the sale of bootleg drugs and counterfeiting. as a result, in 1953 the american pharmaceutical association (apha), the pharmacists ' professional association, and the pharmaceutical manufacturers association (pma) were instrumental in establishing state antisubstitution laws as a means of preventing distribution of drug products that were designed to look like brand - name products but were not, so called, counterfeiting. in april of 1970, however, the apha reversed its stand and advocated repeal of the antisubstitution laws. apha argued that counterfeiting no longer existed because of stringent federal control, and that the pricing policies of the drug industry were being designed to take advantage of the antisubstitution laws. moreover, it was argued, pharmacists were in the best position to judge the quality of drug products and, as they were in direct contact with the sources of supply, could lower the cost of prescription drugs by selective purchasing and dispensing (report of the public affairs committee, 1970). the pma, the american medical association (ama), the national association of chain drug stores (nacds), and the national association of retail druggists (nard) all opposed the apha position. the apha then changed its strategy and advocated amending, rather than repealing, state antisubstitution laws. by 1980 california 's antisubstitution law, for example, was amended in 1975 and states that a pharmacist filling a prescription order for a drug product prescribed by its trade name or brand name may select another drug product with the same active chemical ingredients of the same strength, quantity, and dosage.. the amendment continues, in no case shall a selection be made pursuant to this section if the prescriber personally indicates, either orally or in his own handwriting, do not substitute or words of similar meaning. and the person who selects the drug product to be dispensed pursuant to this section shall assume the same responsibility for selecting the dispensed drug product as would be incurred in filling a prescription for a drug product prescribed by generic name. there shall be no liability on the prescriber for an act or omission by a pharmacist in selecting, preparing, or dispensing a drug product. in no case shall the pharmacist select a drug product pursuant to this section unless the drug product selected costs the patient less than the prescribed drug product. a marketing survey was conducted for the purpose of determining what products were actually dispensed when different types of prescriptions were presented to pharmacists. prescriptions were written by cooperating physicians for four major categories of drugs tranquilizer, antibiotic, sulfa - antibiotic, and double - strength sulfa - antibiotic. for drugs in the first category, prescriptions were written either for a generic or for either of two brands. for the second, three brands were included in addition to the generic product. for the last two categories, only two brands of products (no generics) were available. within each category, substitution within each of the drug categories was allowed because the respective products were generically equivalent. the survey was done in four metropolitan areas of california : los angeles, san diego, san francisco, and sacramento. the actual product that was dispensed and the price charged for it were then noted. in the next section, we look at the observed substitution pattern more closely. a pharmacist 's decision to stock and dispense drugs that are available from more than one source of supply depends on many factors. these include the legality of substitution, an assessment of the quality of each manufactured version of the drug, the overall reputation of the manufacturer, the acquisition and inventory cost, the consumer 's ability to pay, the third - party reimbursement policy, the degree of competition in the market in which the pharmacy is located, and the consumer 's and pharmacist 's attitude toward substitution. therefore, the substitution decision is complex and can not necessarily be predicted on the basis of a single factor variable. table 1 shows the substitution pattern for the four categories of drugs included in the marketing survey. pharmacists had the opportunity to substitute within each category if they chose to do so. the data show that the rate of dispensing less expensive brands and generic drugs for more expensive brands is low, below 14 percent of all sampled prescriptions. how can such dispensing patterns be explained, especially in the light of the clear mandate given pharmacists to substitute the less expensive (especially generic) versions of drugs for more expensive brands ? in order to better understand this behavior, we hypothesize and test alternative behavioral decisions on the part of pharmacists. we assume that pharmacies (as other firms) optimize some set of profit - enhancing variables, and so in the following section we will examine various hypotheses about possible objective functions. we begin with the hypothesis that pharmacies maximize their profit margin as they decide what to dispense. this hypothesis suggests that a pharmacy would dispense the drug product with the highest profit margin (pm) whenever possible, within the limits of ethical standards, legal procedure, and generally accepted business practice. profit margin is defined as : pri = dispensed price of the ith drug cdi = acquisition cost of the ith drug dispensed price was observed directly in the marketing survey, but the acquisition cost of each drug product was not. in this study we estimate it by the mean drug acquisition cost reported by the health care financing administration, which administers the maximum allowable cost (mac) program for drug reimbursement for the department of health and human services. the mac cost estimates are not pharmacy specific, but they are the best available reasonably accurate estimates of wholesale costs that pharmacies face. the use of national average acquisition cost may overstate acquisition cost of chain pharmacies while understating it for independent pharmacies. this bias, to the extent that it exists, should apply to all products, whether low or high cost, generic or brand name, and so differences in estimated profitability between drug products will not be affected. differences in profitability across pharmacy type, chain versus independent, however, may be affected. the average price (pr), acquisition cost (cd), and profit margin (pm) for the four categories of drugs are reported in table 2 for chain and independent pharmacies (chain pharmacies refer to the pharmacies that operate in more than one location). the observation of low rates of substitution in favor of less expensive products in table 1 would seem to contradict the maximum profit margin hypothesis, because the profit margin for less expensive brand versions and especially generic drugs is in fact much higher than that for more expensive brand name drugs. this is generally true even though drug acquisition costs vary from one pharmacy to another. a test of the significance of the difference between the average profit margin, pm, of different drugs, brand versus generic, was made. because there are more than two versions of the drugs in the first two categories, tranquilizer and antibiotic, more than one comparison had to be made within those categories. therefore, to test the hypothesis that profit margin of generic drugs was greater than that of brand name versions, a simultaneous multiple technique was used (dunn and clark, 1974). the average profit margin of generic drugs is significantly larger than that of each of the brand name drugs (and their average). therefore the profit margin hypothesis can not explain the substitution pattern observed in table 1. second, maximizing the profit margin (profit rate) would maximize total profit if pharmacies could influence the demand for each category of drugs through their dispensing pattern by, for example, increasing the total number of prescriptions written for each category of drug. but physicians determine the total number of prescriptions written for each category of drug, and so it is the absolute profit, ap, instead, which might be maximized with ap defined as api = pricdi. to remedy the later problem a new version of profit maximization hypothesis is developed. according to this version, pharmacies dispense drug products with the highest absolute profit whenever possible in order to maximize their total profit. although the absolute profit of generic drugs is higher than that for brand products, the difference was not statistically significant at the 1-percent significance level. all of the t -statistics for differences in absolute profit, ap, are insignificant at the 1-percent level. the contrast with the t -statistics for the profit margin, pm, is striking. thus the absolute profit version of the profit maximization hypothesis can not be rejected and the observed dispensing pattern is shown to be consistent with the absolute profit maximization hypothesis. when a pharmacist dispenses a drug, the choice as to which version of the product is to be dispensed has, in one respect, already been made. inventory costs are one of the factors that influence pharmacies ' dispensing patterns. in a state with antisubstitution laws, maintaining a large inventory would be mandatory for pharmacies in order to avoid losing sales for multiple - source products, but maintaining an inventory that includes a large number of different versions of the same drug product is costly. if substitution is allowed, pharmacies have the chance to store fewer versions of products, maybe only one version, and reduce inventory cost substantially. reduction in inventory costs because of substitution was noted by coward (1976) in his study of michigan pharmacies. the argument for the existence of economies of scale in storing and dispensing drug products is supported by cady (1975). the design of the sample in the marketing survey, however, does not allow us to test this hypothesis. this hypothesis suggests that a pharmacy would dispense the drug product with the highest profit margin (pm) whenever possible, within the limits of ethical standards, legal procedure, and generally accepted business practice. profit margin is defined as : pri = dispensed price of the ith drug cdi = acquisition cost of the ith drug dispensed price was observed directly in the marketing survey, but the acquisition cost of each drug product was not. in this study we estimate it by the mean drug acquisition cost reported by the health care financing administration, which administers the maximum allowable cost (mac) program for drug reimbursement for the department of health and human services. the mac cost estimates are not pharmacy specific, but they are the best available reasonably accurate estimates of wholesale costs that pharmacies face. the use of national average acquisition cost may overstate acquisition cost of chain pharmacies while understating it for independent pharmacies. this bias, to the extent that it exists, should apply to all products, whether low or high cost, generic or brand name, and so differences in estimated profitability between drug products will not be affected. differences in profitability across pharmacy type, chain versus independent, however, may be affected. the average price (pr), acquisition cost (cd), and profit margin (pm) for the four categories of drugs are reported in table 2 for chain and independent pharmacies (chain pharmacies refer to the pharmacies that operate in more than one location). the observation of low rates of substitution in favor of less expensive products in table 1 would seem to contradict the maximum profit margin hypothesis, because the profit margin for less expensive brand versions and especially generic drugs is in fact much higher than that for more expensive brand name drugs. this is generally true even though drug acquisition costs vary from one pharmacy to another. a test of the significance of the difference between the average profit margin, pm, of different drugs, brand versus generic, was made. because there are more than two versions of the drugs in the first two categories, tranquilizer and antibiotic, more than one comparison had to be made within those categories. therefore, to test the hypothesis that profit margin of generic drugs was greater than that of brand name versions, a simultaneous multiple technique was used (dunn and clark, 1974). the average profit margin of generic drugs is significantly larger than that of each of the brand name drugs (and their average). therefore the profit margin hypothesis can not explain the substitution pattern observed in table 1. second, maximizing the profit margin (profit rate) would maximize total profit if pharmacies could influence the demand for each category of drugs through their dispensing pattern by, for example, increasing the total number of prescriptions written for each category of drug. but physicians determine the total number of prescriptions written for each category of drug, and so it is the absolute profit, ap, instead, which might be maximized with ap defined as api = pricdi. to remedy the later problem a new version of profit maximization hypothesis is developed. according to this version, pharmacies dispense drug products with the highest absolute profit whenever possible in order to maximize their total profit. although the absolute profit of generic drugs is higher than that for brand products, the difference was not statistically significant at the 1-percent significance level. all of the t -statistics for differences in absolute profit, ap, are insignificant at the 1-percent level. the contrast with the t -statistics for the profit margin, pm, is striking. thus the absolute profit version of the profit maximization hypothesis can not be rejected and the observed dispensing pattern is shown to be consistent with the absolute profit maximization hypothesis. when a pharmacist dispenses a drug, the choice as to which version of the product is to be dispensed has, in one respect, already been made. inventory costs are one of the factors that influence pharmacies ' dispensing patterns. in a state with antisubstitution laws, maintaining a large inventory would be mandatory for pharmacies in order to avoid losing sales for multiple - source products, but maintaining an inventory that includes a large number of different versions of the same drug product is costly. if substitution is allowed, pharmacies have the chance to store fewer versions of products, maybe only one version, and reduce inventory cost substantially. reduction in inventory costs because of substitution was noted by coward (1976) in his study of michigan pharmacies. the argument for the existence of economies of scale in storing and dispensing drug products is supported by cady (1975). the design of the sample in the marketing survey, however, does not allow us to test this hypothesis. it has been widely argued that prescribing and dispensing generics has a vast saving potential for consumers (borok and schweitzer, 1979). the federal trade commission (1979) staff estimated that total savings at the wholesale level could have been $ 817 million in 1977 had subsitution possibilities been fully utilized and the lowest price generic products substituted for all brand name prescriptions written. in a study of drug substitution in michigan, goldberg (1978) reported a saving of 65 per generic drug written and dispensed. however, when prescriptions were written for brand name products but generic products were dispensed, the saving per prescription was $ 1.14. in this section, different pharmacy pricing formulae, or models, are estimated in order to examine the influence of the substitution laws. the pricing formulae are then used to test the hypothesis that pricing is different when a substitute drug is dispensed than when the ordered drug is dispensed. further, one can use these pricing formulae to examine the extent to which potential savings are passed on to consumers. we assume that the supply of each and every drug to each and every pharmacy is perfectly elastic, that is, the acquisition cost of all drugs is fixed and is the same for all pharmacies. the total amount of demand, that is, the number of prescriptions written for each and every category of prescribed drug (e.g., antibiotics), is exogenously determined by physicians. however, consumers are price sensitive and, therefore, there will be competition among pharmacies. the competition among pharmacies, chain versus independent, would influence their pricing behavior and is reflected in the professional fee and the markup. the difference in acquisition cost (dac) of what was ordered and what was dispensed, could be positive (substitution in favor of a less expensive product), zero (no substitution), or negative (substitution in favor of a more expensive product). this leads us to the following model : the coefficients a0, a1, a2, and a3 define the professional fee under different circumstances, and a4 through a7 define the markup. are professional fees and/or markups affected by the decision to substitute ? and finally, do independent pharmacies charge higher professional fees and/or markups ? an examination of the data provided by the survey suggested the presence of heteroskedasticity in the model with the variances of the error term uijk not being equal across all settings and drugs. use of bartlett 's test (intriligator, 1978) confirmed our suspicion, because the value of chi - square was 78.46 which is significant at the 1-percent level. bartlett 's test for nonhomogeneity of variances, q / l, has a chi - square distribution with p1 degree of freedom. this implies that the use of ordinary least squares yields inefficient estimators. to estimate equation 1 in the presence of heteroskedasticity, the residuals obtained from the first round of ordinary least squares estimates (column 1 in table 4) the estimate of variances is then used as weights to estimate equation 1 in a second round using weighted least squares (kmenta, 1971). the iterative procedure was discontinued when the estimates converged after the seventh round of estimation. the final results of equation 1, when simplified, are presented in table 5 (part a). first, pharmacies do charge a professional fee ($ 2.28) as well as a markup, and the professional fee is 81 higher for independent pharmacies ($ 3.09) than for chains. this could be attributed to a wider range of services generally provided by independent pharmacies or to economies of scale enjoyed by chains. the markup charged by independent pharmacies is 4 percent lower than that charged by chains, however, the difference in the markup is not statistically significant. if the common acquisition cost assumption introduces a bias, as discussed earlier, the observed difference in markup between pharmacy types will be understated, and the difference between professional fees will be overstated. second, the professional fee and the markup are both affected by the decision to substitute. the professional fee and the markup increase by 73 and 2 percent respectively, as pharmacies substitute in favor of less expensive drugs, e.g., generics. the finding is consistent with the findings of schwartzman (1976) who reported higher markups for generic dispensing. when pharmacies substitute in favor of more expensive drugs the professional fee drops by 88 but the markup increases by 18 percent. as long as the difference in price of what is ordered and what is dispensed is less than $ 4.90, the net effect is a reduction in price of drug dispensed. it might be argued that the only legitimate form of substition is the substitution of less expensive drugs for more expensive ones. to investigate this, equation 1 is reestimated using the same technique as before, with all the cases for which substitution was in favor of more expensive drugs treated as no substitution. the results are reported in columns 3 and 4 of table 4 and are summarized in part b of table 5. the findings do not change, as the professional fee is observed to be 86 higher in independent pharmacies than in chain pharmacies, and is 96 higher when pharmacies substitute in favor of less expensive drugs. the markup is 4 percent lower for independent pharmacies than for chains and is 2 percent lower when substitution takes place. now we turn to the question of potential savings to consumers. the california substitution law states the pharmacist shall pass on to the purchaser the difference in the acquisition cost between the drug product prescribed and the drug product dispensed, exclusive of the pharmacist 's professional fee. the pharmacist may not charge a higher or different professional fee for the generic drug product dispensed than that charged for the brand name product prescribed although the law is specific about the professional fee, it does not mention anything about the markup. therefore, pricing of the drugs, in case of substitution, and the amount of saving that should be passed on to the purchaser are left unspecified. the first interpretation of the law is that pharmacies should maintain the same pricing formula regardless of whether or not substitution is made. in such case d1 and d2, substitution dummy variables, thus, if a generic drug is substituted for a brand name product, the pharmacist should price that generic product at the same cost as if it were a brand name product. ff represents the single pricing formula used by a pharmacist to price drug products regardless of the kind of drug dispensed, brand or generic, and the nature of substitution made. for example, a drug that costs the pharmacists oc1 will be priced op1. the price charged has two components c1a(= oc1) the acquisition cost and ap the absolute profit made by pharmacists. ap in turn has two components, ab(= of) the professional fee and bp the amount of markup. however, if pharmacists substitute a different product, say a generic product, that costs only oc2, then its price would be op2, using the same pricing formula ff. the saving that will be passed on to the consumer from substitution is p1p2 which is equal to sum of de (= pd = c2c1), the difference in acquisition cost, and ep, a part of markup that now is passed on to the consumer. the absolute profit made by pharmacists will be ap, which is smaller than ap as shown in figure 1. in terms of equation 1, the price, pr ; the savings that should be passed on to consumer, s ; and absolute profit made per prescription, ap, will be : where cd is the acquisition cost of the drug dispensed. our estimates of equation 1 indicate that both the professional fee and the markup are significantly affected by the decision to substitute (column 2 of table 4). when substitution is defined as substitution in favor of less expensive drugs (column 4 of table 4), then only the professional fee is significantly affected by the decision to substitute. the second interpretation of the law is that pharmacies are allowed to keep constant the amount of absolute profit they make (presumably at the brand name level), and pass on to the consumer only the difference in acquisition cost. in this case, the difference in acquisition cost of what is ordered and what is dispensed, dac, enters directly into the pricing formula. we continue to make the same assumption about the pricing formula as in the case of equation 1. dac, a continuous variable measured in dollars, is used instead of d1 and d2, the two dummy variables in model 1, in order to take into account the substitution decision and to facilitate the test of the hypothesis concerning our second interpretation of the law. ff represents the pricing formula used by a pharmacist when drug products are dispensed as ordered. as in the previous case, a drug product that is dispensed as ordered and which costs the pharmacist oc1 however, if a substitution is made to a generic product that costs the pharmacy oc2, the generic product dispensed would be priced in such a way that the absolute profit made by the pharmacist remains constant, and so the generic drug will be priced op3. the absolute profit made by a pharmacist will be ap, equal to ap, the absolute profit made by a pharmacist if the brand name product ordered had been dispensed. the savings that will be passed on to the consumer will be p1p3, which is less than p1p2, the savings that would have been passed on to the consumer if the pharmacist had used the same pricing formula, ff. in terms of equation 2, price pr ; the savings that should be passed on to the consumer, s ; and the absolute profit made per prescription, ap, will be when co is the acquisition cost of the drug ordered. the constant profit hypothesis, our second interpretation of the law, requires that (b1b2 + b4d3b5d31) = 0. the results are reported in columns 1 and 2 of table 6 and are summarized in part a of table 7. the results confirm the previous findings about the existence of a professional fee as well as a markup and that independent pharmacies charge a higher professional fee, $ 1.11. although the markup by independent pharmacies is 1 percent lower than that of chain pharmacies, their indirect markup, the coefficient of dac in equation 2, is 10 percent higher. but neither of these differences in the markup are statistically significant. therefore, it is indeterminant as to which type of pharmacies pass on a greater portion of acquisition cost savings when substitution takes place. our second interpretation of the law, the constant profit hypothesis, as discussed earlier, implies b1b2 = 1 for chains and (b1 + b4) (b2 + b5)) = 1 for independent pharmacies. to test this hypothesis we used an f test for the difference of coefficients estimated from weighted least squares. the results indicate that the constant profit hypothesis could be maintained for both chain and independent pharmacies because the values of the f statistics were 0.44 and 0.04, respectively. the hypothesis that the difference in the markup and indirect markup is equal to 1 was not rejected. in short, estimates of the pricing formula suggest that pharmacies do price drugs differently when a substitution is made and that pricing aims to maintain a constant absolute profit. it appears that differences in acquisition cost of drug products ordered and dispensed are passed on to consumer. as we did for equation 1, we next estimated equation 2 setting all differences in acquisition costs for cases in which substitution in favor of a more expensive drug was made to zero. results are reported in columns 3 and 4 of table 6 and summarized in part b of table 7. however, the f statistics to test the constant profit hypothesis becomes significant, at a 1-percent level, 4.02 and 4.62 for chain and independent pharmacies respectively, suggesting that the constant profit hypothesis should be rejected. the relative size of the coefficients indicate that pharmacies pass on to consumers an amount less than the difference in the acquisition costs of drug product ordered and drug product dispensed, when substitution is defined as dispensing only lower cost products. attempts to contain the costs of prescribed drugs have taken many forms, one of which is the stimulation of market competition for drugs on which protection has expired. actual savings realized by this policy have been far less than initially expected for a number of reasons, including the relatively low rate of substitution. in an attempt to explain low rates of substitution for a sample of drugs frequently used among california pharmacies, alternative models of economic behavior and profit maximization were tested. the drug substitition issue is complex, involving not only physician preferences for different brands or generic products, but pharmacist cooperation in an area that threatens professional prerogatives and economic performance. a frequently cited reason for the reluctance of both physicians and pharmacists to prescribe and dispense generic drugs is the concern over lack of appropriate safeguards on the quality and efficacy of different products. this concern must be addressed in order for drug substitution to expand to the point of offering substantial cost savings in the health system. this complexity led us to examine a number of economic models in an attempt to explain observed substitution patterns. if pharmacies sought to maximize the profit margin, they would have substituted more than observations indicated. observed patterns of dispensing do appear consistent with the hypothesis that pharmacies attempt to maximize absolute profit per prescription, which may be consistent with overall profit maximization under the assumption that the demand for the product is determined exogeneously by the physician rather than by the pharmacy itself. consistent with this notion is the recognition that inventory costs may play an important role in pharmacist decisionmaking because multiple - source drugs are generally available in a large number of forms, making a full inventory of all available versions of the same drug impractical. our analysis of the pricing formula gives useful insights into the pricing decision of pharmacists, with regard to the use of a professional fee, as opposed to an ordinary percentage markup, and the relationship between profit margin and acquisition cost. the professional fee tends to be higher in independent pharmacies than in chain pharmacies, but the markup is the same across pharmacy type. both the professional fee and the markup are higher when a substitution is made in favor of a less expensive product, so that the absolute profit produced by dispensing a brand - name or generic drug is the same. the highest fee appears to be charged by independent pharmacies when they substitute, and the lowest by chain pharmacies dispensing as ordered. whether or not savings as a result of substitution are passed on to consumers is a more difficult question than might be presumed because there are many definitions of savings. furthermore, we observe that the professional dispensing fee associated with substitution in favor of less expensive products in general, and generic products in particular, exceeds that for brand - name drugs. what does appear to be the case, however, is that substitute drugs are priced so as to yield approximately the same absolute profit, and so the cost differentials associated with the substitute drugs are largely passed on to consumers.	one of the major directions of health policy is the attempt to contain expenditures on pharmaceuticals by encouraging substitution of generic for brand name drug products. yet, a major marketing survey of prescribing and dispensing patterns in california in 1977 found relatively little drug substitution occurring, and in fact substitution of more expensive products occurred more frequently than did substitution of less expensive products.this article tests alternative models of pharmacy dispensing behavior to better explain substitution patterns and it estimates price functions to measure the extent to which cost savings on generic products are passed on to consumers.
a 65-year - old male patient who presented with right arm weakness revealed an acute focal cortical ischemic change in the borderzone type on the diffusion - weighted image (not shown). driver (medtronic ireland, minneapolis, mn) with 2.25 mm diameter and 8 mm length were deployed to 2.35 mm diameter at 12 atmosphere of balloon pressure. our institutional review board approved this study, and we obtained written informed consent from the patient. three - dimensional (3d) rotational angiography was obtained in axiom artis zee (siemens, erlangen, germany) and then the images were transferred to syngo workstation (version vb 15d) to generate 3d angiography with reducing fov and adjusting the target vessel. the final image was transformed to stl format in 256 256 pixels. after smoothing of vessel surface and trimming of branch vessels in magics rp (materialise, belgium),, we carried out cfd and fully coupled fsi simulations with an assumption of rigid wall and compliant wall, respectively. blood flow was assumed to be newtonian, homogeneous flow with density 1050 kg / m, viscosity of 3.5 10 pas. the si physical unit of dynamic viscosity is the pascalsecond (pas), (equivalent to ns / m, or kg/(ms)). to achieve true boundary conditions corresponding with patient - specific, flow velocity before and after installed stent were obtained by transcranial doppler ultrasounds method from gated phase contrast mr angiography in an age - matched male. pulsatile flow rates waveform based on in vivo measurement data was specified at the posterior communicating artery, the anterior cerebral artery and the middle cerebral artery for pre- and post - stent simulations respectively and time - varying pressure condition according to perktold. was imposed in the internal carotid artery inlet. in simulations with compliant wall model, intracranial arterial wall and plaque components were assumed to be isotropic, linear, elastic with a constant young modulus e = 2.6 10 pa, density of p = 1000 kg / m and a poisson 's ratio v = 0.49. to simplify the complex stent geometry, stent was modeled as shell type tube with mechanical property of density e = 2.2 10pa, p = 8420.39 kg / m and possion 's ratio v = 0.234 (co - cr alloy, astm f563).. computational analysis of blood flow in intracranial blood vessels was performed using the commercial finite element software adina version 8.7.2 (adina r & d, inc., fluid domain was divided into 525,659 tetrahedral elements and elastic wall discretization yielded 77,264 shell elements for pre - stent case. because of rigorously complicated flow field through stenosis, dense mesh with gradual ratio change from normal to lesion region was required and especially computational mesh had the highest density around stenosis region. in case of post - stent model, 354,009 tetrahedral fluid elements and 55,359 shell elements were used for fluid and structure domain respectively. in order to prevent torsion and rotation of vessel, constraints at intracranial ends were placed and rotation motions around global coordinate axis system were fixed. we compared difference of wss before and after stenting regarding to maximum wss at systole and enddiastole. a 65-year - old male patient who presented with right arm weakness revealed an acute focal cortical ischemic change in the borderzone type on the diffusion - weighted image (not shown). driver (medtronic ireland, minneapolis, mn) with 2.25 mm diameter and 8 mm length were deployed to 2.35 mm diameter at 12 atmosphere of balloon pressure. our institutional review board approved this study, and we obtained written informed consent from the patient. three - dimensional (3d) rotational angiography was obtained in axiom artis zee (siemens, erlangen, germany) and then the images were transferred to syngo workstation (version vb 15d) to generate 3d angiography with reducing fov and adjusting the target vessel. the final image was transformed to stl format in 256 256 pixels. after smoothing of vessel surface and trimming of branch vessels in magics rp (materialise, belgium), in the current study, we carried out cfd and fully coupled fsi simulations with an assumption of rigid wall and compliant wall, respectively. blood flow was assumed to be newtonian, homogeneous flow with density 1050 kg / m, viscosity of 3.5 10 pas. the si physical unit of dynamic viscosity is the pascalsecond (pas), (equivalent to ns / m, or kg/(ms)). to achieve true boundary conditions corresponding with patient - specific, flow velocity before and after installed stent were obtained by transcranial doppler ultrasounds method from gated phase contrast mr angiography in an age - matched male. pulsatile flow rates waveform based on in vivo measurement data was specified at the posterior communicating artery, the anterior cerebral artery and the middle cerebral artery for pre- and post - stent simulations respectively and time - varying pressure condition according to perktold. was imposed in the internal carotid artery inlet. in simulations with compliant wall model, intracranial arterial wall and plaque components were assumed to be isotropic, linear, elastic with a constant young modulus e = 2.6 10 pa, density of p = 1000 kg / m and a poisson 's ratio v = 0.49. to simplify the complex stent geometry, stent was modeled as shell type tube with mechanical property of density e = 2.2 10pa, p = 8420.39 kg / m and possion 's ratio v = 0.234 (co - cr alloy, astm f563). computational analysis of blood flow in intracranial blood vessels was performed using the commercial finite element software adina version 8.7.2 (adina r & d, inc., fluid domain was divided into 525,659 tetrahedral elements and elastic wall discretization yielded 77,264 shell elements for pre - stent case. because of rigorously complicated flow field through stenosis, dense mesh with gradual ratio change from normal to lesion region was required and especially computational mesh had the highest density around stenosis region. in case of post - stent model, 354,009 tetrahedral fluid elements and 55,359 shell elements were used for fluid and structure domain respectively. in order to prevent torsion and rotation of vessel, constraints at intracranial ends were placed and rotation motions around global coordinate axis system were fixed. we compared difference of wss before and after stenting regarding to maximum wss at systole and enddiastole. the wall shear stress distributions from cfd simulation with rigid wall assumption as well as fsi simulation before and after stenting were compared in fig. overall, the difference of wss between rigid wall and compliant wall model both in pre- and post - stent case is only minor except at the stenosis region. in the case of pre - stenting, fsi simulation created noticeable reduction of maximum wss at the stenosis compared to the simulation with rigid wall assumption. before stent implantation, maximum wsss at the stenosis were calculates as 167 pa at systole and 109 pa at end - diastole in cfd simulation with rigid wall assumption and as 138 pa at systole and 104 pa at end - diastole in fsi simulation with considering wall compliance (fig. these wss values were greatly reduced after stenting to 15~20 pa at systole and 3~5 pa at end - diastole in cfd simulation, which are similar in fsi simulations. this revealed that the structural stress at the stenosis region is not rather high compared with the one on normal vessel, but in similar range even before implanting stent. this implies that wss may play more important role in plaque rupture by fissuring of thin fibrous cap than structural stress on plaque. fluid - structure interaction (fsi) analysis has been employed as a powerful tool because it combines blood flow simulation through cfd with wall tensile stress (wts) analysis in the plaque region by the finite element method. blood flow through a compliant artery requires appropriate fluid - structure coupling in order to account for the interaction between the flowing blood and the deforming arterial wall. with anatomically realistic plaque geometry, fsi simulation is able to provide detailed stress analysis for the plaque which has been demonstrated with individual case studies. as we could demonstrated the difference of fsi compared to cfd without considering the fsi, ultimately it is hoped that biomechanical simulations based on patient - specific data will serve as a reliable and robust clinical tool in the characterization of lesions and choice of treatment plan. although various arterial sites have been analyzed by fsi such as the carotid arterial bifurcation, an abdominal aortic aneurysm model due to relative ease of imaging and simpler physical boundary conditions, and a stented aortic aneurysm model, our results revealed that the application of cfd and fsi can also be possible in the intracranial vessels which has small diameter in the range of 2 - 3 mm [13, 19, 20 ]. risk assessment based solely on the degree of luminal stenosis will tend to underestimate the severity of the atherosclerotic lesion. for these reasons, there is a need for more sensitive methods to risk stratify patients with intracranial atherosclerotic disease. currently, patient - specific 3d fsi simulations suffer from two major bottle necks in terms of the time required to get from medical images to mechanical stress and strain predictions. the first challenge is the generation of a suitable computational mesh that can accurately represent the components of the diseased artery and can provide meaningful numerical results. the second challenge relates to the time and computational resources required to solve realistic finite element models on a patient - specific basis. the two challenges are inter - related, and reduction of one generally means an increase in the other. to estimate stress levels in the fibrous cap, fsi analysis has emerged as a tool combining blood - flow simulation through cfd with finite element analysis of the corresponding stress levels in the surrounding tissues. this study has some limitations. intrinsically, vessel wall is nonlinear hyperelastic material and even its properties vary with locations and the components of vessel, in particular, at the stenosis with plaque. we do note, however, that our finding that wss would be essential in plaque rupture compared to structural stress in the perspective of hemodynamics would hold because the narrower and thicker vessel in stenosis is, the less structural stress, but higher wss would be produced. however, this would be proper assumption in modeling because real stent would be relatively rigid after implantation with outward expanding narrowed vessel. the assumption of newtonian rheology made in this study have also shown to be of relatively minor influence on hemodynamic characteristics in large vessel.	purposeimage - based computational models with fluid - structure interaction (fsi) can be used to perform plaque mechanical analysis in intracranial artery stenosis. we described a process in fsi study applied to symptomatic severe intracranial (m1) stenosis before and after stenting.materials and methodsreconstructed 3d angiography in stl format was transferred to magics for smoothing of vessel surface and trimming of branch vessels and to hypermesh for generating tetra volume mesh from triangular surface - meshed 3d angiogram. computational analysis of blood flow in the blood vessels was performed using the commercial finite element software adina ver 8.5. the distribution of wall shear stress (wss), peak velocity and pressure was analyzed before and after intracranial stenting.resultsthe wall shear stress distributions from computational fluid dynamics (cfd) simulation with rigid wall assumption as well as fsi simulation before and after stenting could be compared. the difference of wss between rigid wall and compliant wall model both in pre- and post - stent case is only minor except at the stenosis region. these wss values were greatly reduced after stenting to 15~20 pa at systole and 3~5 pa at end - diastole in cfd simulation, which are similar in fsi simulations.conclusionour study revealed that fsi simulation before and after intracranial stenting was feasible despite of limited vessel wall dimension and could reveal change of wss as well as flow velocity and wall pressure.
a thorough knowledge of the external and internal anatomy of teeth is a very important factor in root canal treatment. in many cases, dentists have to deal with various morphological variations. if the dentist fails to detect the morphological variations, it would be a major cause of failure. when a preoperative radiograph shows an atypical tooth shape, further radiographic examinations should be considered in order to detect unusual anatomical differences. in maxillary first molars, morphological variations, such as abnormal numbers of roots, canals, fusion and germination and the existence of c - shaped root canals have been widely known. a few cases of c - shaped root canals in maxillary molars have been reported, though c - shaped canals are most frequently found in mandibular second molars. some authors have reported that c - shaped canals result from the fusion of mesiobuccal (mb) and palatal (p) roots of maxillary molars, while others have reported that the distobuccal (db) and p roots of maxillary molars were fused, and even a case of fusion of the mb and db roots of maxillary molars was reported. the incidence of c - shaped canals in maxillary first molar has been reported to be as low as 0.091% based on radiographic examination. in case of anatomical abnormalities, periapical surgery, intentional replantation and even extraction intentional replantation has been performed for more than a thousand years and this technique consists of intentional tooth extraction, cleaning of the apical part of the tooth and reinsertion of the extracted tooth into its own socket immediately. many authors agree that this technique should be the last option ' after all the other procedures have failed or when endodontic periradicular surgery can not be performed. the purpose of this report is to present a morphological variation of c - shaped canal in a maxillary first molar in which the mb, db and p roots were fused to mimic the letter o '. a 39-year - old male was referred by a private practitioner to the department of conservative dentistry at yonsei university dental hospital. the reason for referral was high possibility of fracture of the maxillary left first molar while trying to remove a pre - existing old post in the palatal canal (figure 1). the tooth had been treated endodontically and restored with a post and core 10 years ago. however, he had no symptoms at that point in time. on the clinical examinations, percussion and mobility tests were within normal limits and probing depth was also normal. based on clinical and radiographic findings, the diagnosis of chronic apical periodontitis was established. the possibility of root perforation by the post and sinus involvement by the roots could not be ignored as the cause of the symptom. for further examination cone - beam computed tomography (cbct, rayscan symphony ; ray co., ltd, seoul, cbct examination revealed a single - rooted maxillary first molar, and all the roots seemed to be fused together into one o - shaped root. the sinus wall seemed to be intact (figure 3), but the possibility of perforation by the post or root fracture could not be excluded because it was presumed that the existence of an o - shaped root was unlikely at that time and the overlapping of root images were persistent. it was concluded that the conventional root canal retreatment was not possible because of difficulty in negotiating all canals and the possibility of root fracture during removal of the post. hence, intentional replantation was planned. on the day of surgery, patient received a preoperative regimen of antibiotics and anti - inflammatory drugs. with delicate luxation using a root elevator, the tooth was extracted without fracture. the inflamed granulation tissue in the center of the fused roots was removed meticulously, and one root with an o - shape was observed. on a side view, the root was rectangular in shape, and on an apical view all roots were fully connected and no perforation by the post was observed (figure 4). when the tooth was examined with a surgical operating microscope (carl zeiss opmi pico ; carl zeiss, oberkochen, germany), more than five or six small foramina were observed. it was decided to resect the apical end of the root for removing the unnoticed small foramina. the apical 3 mm of the root were trimmed. on the prepared apical o - shaped root surface there were 56 root canals with connecting fins, and hence, a 360 circular root end cavity was made with an ultrasonic tip and it was checked by methylene blue (figure 5). during intentional replantation, the tooth was kept under wet gauze for maintaining the pdl cells of the root surface vital. the root canal was re - cleaned and filled with retrograde root filling material (resin - modified glass ionomer ; fuji ii ; gc, tokyo, japan) to cover the long root end cavity. and the tooth was re - implanted into its own socket. at the 9 months recall visit, the tooth was asymptomatic and a progressive healing of the lesion was evident (figure 6). we herein present the case of a patient with unusual root morphology of the maxillary first molar which has not been reported up to now. we named this morphological variation as an o - shaped root following the concept of c - shaped roots. at first, we suspected that this tooth had a c - shaped root because a blunt apex and an unclear root shape were seen on the periapical radiographs. the axial and cross - sectional view of the maxillary arch showed the symmetric morphology of the maxillary first molars with an o - shaped root, but we could not exclude the possibility of intimate proximity of roots or simple fusion between the c - shaped root and the other root. at first, periapical microsurgery was considered for establishing the diagnosis and management of the unusual root morphology. however, the tooth also had difficulties with surgical approaches and the possibility of maxillary sinus perforation during microsurgery. as a result, intentional replantation was planned carefully because the possibility of tooth fracture could not be excluded. the extraction and replantation procedure was also expected to be difficult because of the rectangular shape of the root. extraction of the tooth from its socket was done successfully without causing root fracture or alveolar bone fracture, while trying to preserve the periodontal ligament and not exerting too much pressure on the tooth and socket walls. after removal of the granulation tissue that covered the root from the apical concave area up to the normal furcation area, an o - shaped root was observed. no visible perforation by the post was detected on the root surface, and also no sinus involvement was detected. because of this inner granulation tissue, the possibility of perforation by the post could not be excluded. in this case, the extraoral time needed was almost 17 min for meticulous extraction and management of the unusual root morphology. reported that the initial ankylosis did not show when experimental group tooth was treated with the extraoral (complete dry) time of 15 min., mineral trioxide aggregate has been accepted as the material of choice for root - end filling in endodontic surgery, but mineral trioxide aggregate is a technique sensitive material of root end filling for handling in comparison with other materials. in this case, resin - modified glass ionomer was selected as a retrograde filling material because it had marginal sealing ability in narrow root end cavity, though it was less tissue - tolerant. unusual root anatomy of the maxillary molars that has been reported previously includes the fusion of buccal roots, the fusion of mb and p roots, and the fusion of db and p roots, but to the best of our knowledge this is the first case report of all roots being fused together forming an o - shape with a normal furcation. in this case, cbct was useful for diagnosing the unusual root morphology because of its ability to display the serial sections of the tooth. meticulous examination and recognition of an o - shaped root morphology using periapical radiographs and cbct could be helpful to diagnose the rare o - shaped root '. the value of this case report was to present maxillary first molar with a very unusual o - shaped root canal system, as such case is seldom mentioned in textbooks. during endodontic therapy, even though the incidence of an o - shaped root is very rare, the recognition with periapical radiographs and cbct should not be underestimated.	this case report is to present a maxillary first molar with one o - shaped root, which is an extended c - shaped canal system. patient with chronic apical periodontitis in maxillary left first molar underwent replantation because of difficulty in negotiating all canals. periapical radiographs and cone - beam computed tomography (cbct) were taken. all roots were connected and fused to one root, and all canals seemed to be connected to form an o - shape. the apical 3 mm of the root were resected and retrograde filled with resin - modified glass ionomer. intentional replantation as an alternative treatment could be considered in a maxillary first molar having an unusual o - shaped root.
severe acute respiratory syndrome : temporal stability and geographic variation in case - fatality rates and doubling times	we analyzed temporal stability and geographic trends in cumulative case - fatality rates and average doubling times of severe acute respiratory syndrome (sars). in part, we account for correlations between case - fatality rates and doubling times through differences in control measures. factors that may alter future estimates of case - fatality rates, reasons for heterogeneity in doubling times among countries, and implications for the control of sars are discussed.
respiratory distress in a neonate is more commonly caused by pulmonary, cardiac, or neurological etiologies. the obstructive lesions of the upper airway are rare, unsuspected, and potentially treatable causes of neonatal respiratory distress. an oropharyngeal mass presenting as a cause of neonatal respiratory distress is rare and may include bronchogenic cyst, epidermoid cyst, congenital cysts of third and fourth pharyngeal pouches, and the least common basal meningoencephalocele. they originate from a congenital hiatus in the midline region of the skull base, which permits meninges, neural tissue, or both to herniate from the intracranial space. basal encephaloceles occur with an estimated incidence of 1 in 35,000 live births and have been further subdivided, depending on the location of the bone defect, into transethmoidal, sphenomaxillary, sphenoorbital, and transsphenoidal. our patient had presented with respiratory distress and an oropharyngeal mass in the neonatal period and was eventually diagnosed to have transalar transsphenoidal meningoencephalocele. a 30-day - old male infant weighing 2.6 kg presented with difficulty in breathing and feeding noticed since 4 day of life. the infant was observed to have tachypnea with a respiratory rate of 70 breaths per minute along with marked suprasternal retractions and stridor, though cyanosis was absent. arterial blood gas analysis revealed normal oxygenation, and chest x - ray did not show any significant abnormality. the patency of all natural orifices was checked, and choanal atresia, tracheo - esophageal fistula, and anorectal malformation were ruled out. examination of the oropharyngeal cavity showed presence of a cystic swelling protruding through the soft palate. aspiration of the cyst with a 26-gauze needle produced a small quantity of clear fluid with no cells and a protein content of 80 mg% which was compatible with a transudate. subsequently, after 48 hours of aspiration, the child had recurrence of symptoms and reappearance of the swelling. the computed tomographic (ct) scan with bone windows revealed a large defect in the sphenoidal base with herniating meningoencephalocele [figure 1 ]. a transsphenoidal encephalocele was diagnosed on the magnetic resonance imaging (mri) [figure 2 ]. the patient was successfully operated on 40 day of life using the transpalatal approach involving a multidisciplinary team comprising neurosurgeons and otolaryngologists. ct scan shows defect in sphenoid with herniating meningoencephalocele mri showing trans - sphenoidal encephalocele during follow - up on 70 day of life, the child is stable, accepting breast feeds, and neurologically normal. obstructive lesions of the newborn airway include choanal atresia, macroglossia, pierre - robin syndrome, lymphangioma, teratoma or other mediastinal masses, cysts, subglottic stenosis, and laryngotracheomalacia. an oropharyngeal mass presenting as a cause of neonatal respiratory distress is rare and may include bronchogenic cyst, epidermoid cyst, congenital cysts of third and fourth pharyngeal pouches, and the least common basal meningoencephalocele. there are only two case reports in the literature of a newborn presenting with respiratory distress caused by basal transsphenoidal meningoencephalocele. symptoms as a result of a basal encephalocele vary according to the site and size of the lesion. consequently, respiratory distress, episodes of apnea, difficulty with feeding, and failure to thrive can be seen. severe respiratory distress and feeding difficulty as seen in our patient were attributable to pharyngeal airway obstruction. only 11 pediatric cases of transalar transsphenoidal encephaloceles have been reported in the literature, with two of them presenting in neonatal period with respiratory distress [table 1 ]. he was observed to have a cystic mass protruding through the soft palate which was diagnosed on neuroimaging as transalar transsphenoidal meningoencephalocele. pediatric case series of transalar transsphenoidal encephaloceles the majority of transsphenoidal meningoencephaloceles are diagnosed during the first year of life due to manifestations such as respiratory distress caused by epipharyngeal obstruction, feeding difficulties, cranial midline defects with cleft lip or cleft palate, hypertelorism, optic malformations with anophthalmia, retinal abnormalities, optic nerve hypoplasia, unexplained bouts of recurrent meningitis, or endocrine abnormalities. associated congenital anomalies have been noted in one third of the cases of sphenoidal encephalocele. however, if there are no considerable difficulties and no distinctive facial anomalies during childhood, the diagnosis of the disease may be delayed up to adulthood, when distinctive symptoms such as rhinorrhea, visual defect, or endocrine dysfunction occur. advanced imaging studies are necessary to confirm the diagnosis of transsphenoidal encephalocele and to define any neural or vascular elements that may be included in the herniation. ct scan and mri are the most useful modalities for diagnosing meningoencephalocele. in the present case, ct scan including 3d reconstruction allowed visualization of bone defects in the skull base and a well - circumscribed expansile mass lesion in the extracranial area communicating with the intracranial space. mri with gadolinium enhancement evaluated the content of the encephalocele and eliminated other brain anomalies. the contents of the sac need to be preserved as the sac invariably contains vital structures. transsphenoidal encephalocele is frequently accompanied by a split palate, and these conditions can be operated upon at the same sitting via the transpalatal approach. there is also less risk of damaging the functioning tissues within the wall of the encephalocele in transpalatal surgical approach. in conclusion, this case is unique because it concerns an infant with severe respiratory problems from the time of birth as a result of a transsphenoidal encephalocele presenting as an oropharyngeal cystic swelling. obstructive causes of respiratory distress in neonatal period are rare and pose a diagnostic challenge. a detailed oropharyngeal examination should be considered in a neonate with respiratory distress with no apparent cardiac or respiratory cause. it is important to consider early neuroimaging for diagnosing these conditions and plan definitive treatment for successful outcome.	respiratory distress in an infant is a common cause of admission in neonatal intensive care facility. obstructive lesions of the airway constitute a minority of problems in the new born but present a diagnostic challenge. we present a 30-day - old male infant admitted with respiratory distress who was diagnosed to have an oropharyngeal cystic mass which on further evaluation by computed tomography and magnetic resonance imaging revealed a transalar transsphenoidal meningoencephalocele herniating into the oral cavity through a congenital split palate. the patient was operated successfully using a transpalatal approach leading to complete resolution of respiratory distress.
life in a slum environment can have detrimental effects on residents health status, including manifesting as diminished nutritional status. sedentary lifestyles are common in urban centers, and unfortunately, this trend does not exclude slum dwellers.1 although undernutrition had been the main focus of interventions in slum dwellings in the past, a major global shift toward lifestyles conducive to obesity has caused overnutrition to become an issue, as well.2 this phenomenon of having both undernourished and overnourished people living side - by - side is sometimes called the double burden of malnutrition, and much attention is being paid in particular to slum dwelling women s vulnerability to this phenomenon. women are thought to be more economically and nutritionally insecure, and their physical health as mothers is a predictor of the health of offspring. defining population nutritional status epidemiologically is innately problematic. but body mass index (bmi) is a useful first step and is commonly collected in many government - sponsored national health surveys. the threshold of obesity has been internationally accepted as a bmi of 30 kg / m.3 while body adiposity index (bai) and waist circumference are arguably better indices of actual adiposity,4 bmi is still the most commonly used easily applied indices. indeed, with a large population, bmi is both a noninvasive and time efficient measure.5 several studies68 have already attempted to determine the factors that have the greatest impact on south asian women s bmi. pregnant women in assam were found to have a lower bmi if the household consisted of five or more individuals or if income was low.6 according to the national family household survey from 1998 to 1999 (nfhs-2), different administrative states report different average bmis,7 with more developed states, such as punjab, having higher rates of obesity, while less developed states, such as bihar, having higher levels of chronic energy deficiency.7 as well, yadav and krishnan8 found that urbanization itself is a risk factor for a variety of noncommunicable diseases in india, including obesity. with the present study, we analyzed data from the 20052006 indian national family health survey (nfhs-3)9 to determine the demographic and behavioral factors mostly associated with the bmi of indian women living in slums. a previous attempt10 to use these same data to explore bmi categorized their outcome into undernutrition, normal, and overnutrition categories, thus limiting statistical interpretations. in the study described herein, we sought to conduct a more robust multivariate exploration of these same data, controlling for a wider array of putative covariates and retaining the continuous nature of the bmi outcome variable. we believe that this constitutes the first published study of bmi in modern indian slums that features special attention to residents with tribal status. a multiple linear regression was applied to data from the nfhs-3, with demographic and behavioral factors modeled against the continuous outcome of bmi. the nfhs-3 defined slums based on any of the following criteria : 1) based on the designations by the government established by any act including the slum act, 2) governmental recognition of the slum regardless of any previous identification, or 3) under the criterion of a compact area of at least 300 population or ~6070 households of poorly built congested tenements, in unhygienic environment usually with inadequate infrastructure and lacking in proper sanitary and drinking water facilities, which was assessed visually by the surveyor. in this study, we used the slum definition based on the survey supervisor s assessment of slum status based on three criteria mentioned previously. as well, women who indicated they were currently pregnant were excluded since they provide a skewed relationship between demographic factors and bmi. putative covariates showing poor univariate association (p>0.100) with bmi were omitted from inclusion in the final regression model, in order to optimize our model s robustness. ethical approval was received from the research ethics office of the university of ottawa for this study. written informed consent was deemed not required for this study by the research ethics office of the university of ottawa, because it used only retrospective, anonymized patient data. the regression model was determined to be significant (f=9.776, p0.0001), producing an r of 0.206. whether the woman was a member of a scheduled tribe was not found to be significantly associated with changing bmi, but the p - value of that adjusted association was nonetheless quite low (p=0.051). as this is a variable of deep policy relevance, it was useful to conduct an exploratory subanalysis of tribal levels unadjusted association with bmi. results of that analysis showed that tribal women had significantly lower (unadjusted) average bmi than did nontribal women (20.43 vs 22.02, p<0.001). it was further found that those belonging to a tribe were more likely to dwell in maharashtra or andhra pradesh, be in the poorest or poorer wealth index, and to perform manual labor, regardless of bmi (p<0.001). the rise in obesity rates in india has also seen a subsequent increase in noncommunicable diseases, including type 2 diabetes mellitus.11 the naturally strong association between obesity (measured via bmi) and diabetes in the examined sample may affect the relationships of bmi with the other variables. to explore this possibility, the regression was run again without the diabetes variable included. there was no change in covariates statistical significance, thus the diabetes variable was included in the final regression and deemed not to be an effect modifier or a confounding influence. the increasing bmi of slum dwelling women is most significantly and positively associated with the following factors : frequency of watching television ; having diabetes ; increasing age ; higher wealth index ; and living in the states of new delhi, andhra pradesh, and tamil nadu. belonging to a tribe, while not statistically significant in the adjusted model, was an interesting variable in that it showed significant unadjusted association with living in maharashtra or andhra pradesh, doing manual labor, and being in the poorer or poorest wealth index. our major findings are reflected in the existing literature, which confirm that among low income indians, a sedentary lifestyle, including watching television, is a major contributor to obesity and other noncommunicable diseases.12 it is important to note that the analyzed data are from 2005 to 2006. however, these data remain the most comprehensive set describing the entirety of the indian slum dwelling experience and must therefore suffice for the time being. surprisingly, we did not find a significant adjusted association between education level and bmi (given that education is associated with career opportunities and therefore wealth), though the unadjusted relationship was indeed significant (p=0.037). this suggests that other factors may have been mediating the independent relationship between these two factors. this leads us to believe that having assets and adequate housing (as measured by the wealth index) is more influential than educational status alone, however attained. both education and wealth are dimensions of socioeconomic status, which also include some measurement of social class. and in india, there is no more profound indicator of social class than caste. caste classification is long known as an important consideration for doing any sort of population research in india, with hereditary classification of lower ranked individuals associated with a variety of economic, and thus health, disadvantages.13 caste, defined by family lineage, defines a social hierarchy among individuals, often dictating profession, diet, and living conditions. tribal populations are not part of the formal historic caste structure, but tribal peoples have long faced discrimination similar to that experienced by members of the lowest ranked castes.13 to our knowledge, the present results represent the first attempt to model the influence of tribal status on nutritional health within modern slums. while tribe membership accounts for only a small fraction of the individuals in this data set, tribe populations nonetheless constitute a largely socioeconomically disadvantaged group worthy of policy attention. the literature is barren with respect to the health status and needs of tribal peoples living in india s urban centers. their needs represent an important future research direction, given tribal peoples well - documented experiences with marginalization and poverty.14 our main methodological limitation is that presented by challenges in variable coding. caste is a religious classification system mostly used by hindus,13 but limitedly present in some other religions, as well, including sikhs and buddhists. however, of the 7952 women examined, only 68.2% were hindu, over 95% identified as belonging to a caste, despite it being an overwhelmingly hindu designation. it is unclear whether nonhindus reported believing themselves to be part of this hierarchy or whether the question was asked of them poorly. so - called unidentified slums, which are urban areas with slum - like conditions, which nevertheless do not have the formal designation, have similar, if not worse, conditions than identified slums.1 our sample therefore undersamples indians living in the urban conditions we sought to examine. it would be problematic to draw comparisons from our sample to nonslum dwellers, given that many of those may in fact experience similar slum - like conditions without the mantle of a formal slum designation. unidentified slums are known to have similar, if not worse, living conditions than identified slums ; comparatively inferior conditions are related to the fact that unidentified slums are not eligible for publicly funded amenities due to their lack of classification. this reality serves to keep slum dwellers from unidentified slums, including those who may belong to a tribe, in a lower wealth index compared to slum dwellers from identified slums.15 one clear message arising from these data is that slum dwellers in india are not a homogenous population. factors such as basic housing and sanitation contribute to the wealth index calculation and are important to examine in slum dwellers because a need for infrastructure improvement is still prevalent. however, while slum dwellers have largely been characterized as profoundly impoverished, in these data, it was revealed there are slum dwellers belonging to the middle and richer wealth indexes, indicating that certain slum dwellers experience significantly greater economic standing than others. this is a critical consideration in the development of interventions or for providing resources because slum dweller needs will vary ; some segments of the population may already have significant assets available to them while many others require some form of financial assistance. it will be important for future research to analyze the economic characteristics of the slum population under study before an appropriate intervention is formulated. it is important to note that many previous studies have drawn attention to the so - called double burden of under- and overnutrition in india. slum dwellings, which were historically associated with undernutrition, are now experiencing growing rates of obesity due presumably to the lifestyle associated with increasing urbanization.16,17 thus, slum dwellings, like indian urban centers in general, are experiencing the double burden in lock step with the rest of the country. the extent to which slum dwelling may deserve a unique designation that distinguishes such neighborhoods from other urban environments at least for population health purposes may be overstated. while expected factors, such as age, diabetes, and a sedentary lifestyle, are associated with increasing bmi among slum dwelling indian women, the important insight arising from our study is that nutritional health challenges to indian slums may not be dissimilar to challenges experienced by other urban residents, though the experiences of tribal peoples are deserving of more focused attention in future research projects.	backgroundurbanization is increasing around the world, and in india, this trend has translated into an increase in the size of slum dwellings whose environments are suspected of being associated with poor health outcomes, particularly those relating to women s nutritional status. with this study, we sought to determine the factors associated with indian women s body mass index (bmi) in slum environments, with special attention paid to women with tribal status.methodsa multiple linear regression analysis was performed on data from the indian national family health survey (20052006), modeling demographic and behavioral factors suspected of being associated with bmi, with additional focus on the measures of social class, specifically caste and tribal status.resultsincreasing bmi is significantly and positively associated with frequency of watching television, having diabetes, age, wealth index, and residency status in the areas of new delhi, andhra pradesh, or tamil nadu.conclusionalthough belonging to a scheduled tribe was not associated with changes in bmi, unadjusted rates suggest that tribal status may be worthy of deeper investigation. among slum dwellers, there is a double burden of undernutrition and overnutrition. therefore, a diverse set of interventions may be required to improve the health outcomes of these women.
bernstein (1967) identified the degrees of freedom problem the notion that the large number of independently controllable movement system df poses a computational burden to the cns (turvey. bernstein 's solution (see also gelfand and tsetlin, 1966 ; turvey., 1978 ; tuller., 1982) was that rather than controlling each df separately, the df are coupled to form a synergy, enabling the df to regulate each other. this reduces the need to control each df, and allows compensation for variability in one component of the synergy by another. is expressed in figure 1 (kay, 1988 ; riley and turvey, 2002). df that potentially are independent are coupled so that the synergy has fewer df (possesses a lower dimensionality) than the set of components from which it arises. the behavior of the synergy has even fewer df, a second level of dimensional compression as one moves from structural components to the behavior enacted by the interactions among the df. dimensional compression at both stages results from imposing constraints, which couple components so they change together rather than independently. dynamical systems approaches have emphasized dimensional compression (kugler., 1980 ; kugler and turvey, 1987 ; kay, 1988 ; turvey, 1990 ; turvey and carello, 1996). dimensional compression occurs in self - organization (nicolis and prigogine, 1977) and features prominently in synergetics (haken, 1983), a theory of self - organization that describes how systems of many non - linearly interacting, micro - scale components exhibit low - dimensional spatio - temporal patterns. interactions among micro - components give rise to the macroscopic pattern, which then constrains the behavior of the micro - components to sustain the pattern. coordination dynamics (kelso, 1995) applies synergetics to describe how micro - scale neuromuscular processes give rise to macroscopic movement patterns. in interlimb rhythmic coordination (synchronized oscillations of body segments, such as the rhythmic patterns of walking legs), the order parameter relative phase (the difference in the segments oscillation phases) captures the low - dimensional behavior that arises from the high - dimensional neuromuscular system. relative phase describes the spatiotemporal pattern of rhythmic coordination and the changes in coordination that occur in response to manipulations of control parameters (e.g., movement frequency). the dynamics of relative phase are understood to reflect the behavior of a synergy (kelso, 1994 ; turvey and carello, 1996). dimensional compression is a necessary but insufficient condition for the existence of a synergy (latash, 2008). the second and more critical characteristic of synergies, reciprocal compensation, refers to the ability of one component of a synergy to react to changes in others. a classic example occurs when one effector is perturbed during speech (kelso., 1984). when the lower jaw was tugged downward, it was quickly compensated by a reciprocal change (the lower lip extended upward) that enabled the speaker to complete pronunciation of the sound. reciprocal compensation is central to the uncontrolled manifold (ucm) approach (scholz and schner, 1999 ; latash., 2002). this approach assumes that coordinated movement is achieved by stabilizing the value of a performance variable (such as a value of relative phase corresponding to an interlimb coordination pattern). in doing so, a subspace (i.e., manifold) is created within a state space of task - relevant elements (the df that participate in the task, for example the angular positions and velocities of two oscillating limbs), such that within the subspace the value of the performance variable remains constant. component values that do not lead to desired values of the performance variable (values outside the ucm) are restricted, whereas values that do not affect the performance variable (those within the ucm) are allowed.., 2002) is using two fingers to produce a total force of, for example, 10 n. this target can be achieved by producing 5 n of force with each finger, or by pressing unequally hard with the fingers but so that the total force is 10 n (e.g., one finger produces 9 n and the other produces 1 n, or one produces 7 n while the other produces 3 n, etc.). if the force produced by each finger is plotted on orthogonal axes, the ucm is a subspace of the resulting plane, a line corresponding to forcefinger 1 + forcefinger 2 = 10 n. as long as performance falls along the ucm, the target force is achieved as one finger compensates for the other. in the ucm analysis the hypothesized stabilization of a performance variable is evaluated by computing two quantities. the first, variance along the ucm, measures the extent to which variability among the df is compensated to preserve the performance variable. variance perpendicular to the ucm measures uncompensated variability that causes the performance variable to deviate from the target. while the individual variance components are often informative, their ratio provides a compact measure of the existence and strength of a synergy. if compensated variance is greater than uncompensated variance (ratio > 1), the hypothesized performance variable is stabilized by compensation among the df the higher the ratio, the greater the amount of compensated variance, suggesting a stronger synergy (depending on the magnitude of uncompensated variance). 1987) is expressed in figure 1 (kay, 1988 ; riley and turvey, 2002). df that potentially are independent are coupled so that the synergy has fewer df (possesses a lower dimensionality) than the set of components from which it arises. the behavior of the synergy has even fewer df, a second level of dimensional compression as one moves from structural components to the behavior enacted by the interactions among the df. dimensional compression at both stages results from imposing constraints, which couple components so they change together rather than independently., 1980 ; kugler and turvey, 1987 ; kay, 1988 ; turvey, 1990 ; turvey and carello, 1996). dimensional compression occurs in self - organization (nicolis and prigogine, 1977) and features prominently in synergetics (haken, 1983), a theory of self - organization that describes how systems of many non - linearly interacting, micro - scale components exhibit low - dimensional spatio - temporal patterns. interactions among micro - components give rise to the macroscopic pattern, which then constrains the behavior of the micro - components to sustain the pattern. coordination dynamics (kelso, 1995) applies synergetics to describe how micro - scale neuromuscular processes give rise to macroscopic movement patterns. in interlimb rhythmic coordination (synchronized oscillations of body segments, such as the rhythmic patterns of walking legs), the order parameter relative phase (the difference in the segments oscillation phases) captures the low - dimensional behavior that arises from the high - dimensional neuromuscular system. relative phase describes the spatiotemporal pattern of rhythmic coordination and the changes in coordination that occur in response to manipulations of control parameters (e.g., movement frequency). the dynamics of relative phase are understood to reflect the behavior of a synergy (kelso, 1994 ; turvey and carello, 1996). dimensional compression is a necessary but insufficient condition for the existence of a synergy (latash, 2008). the second and more critical characteristic of synergies, reciprocal compensation, refers to the ability of one component of a synergy to react to changes in others. a classic example occurs when one effector is perturbed during speech (kelso., 1984). when the lower jaw was tugged downward, it was quickly compensated by a reciprocal change (the lower lip extended upward) that enabled the speaker to complete pronunciation of the sound. reciprocal compensation is central to the uncontrolled manifold (ucm) approach (scholz and schner, 1999 ; latash., 2002). this approach assumes that coordinated movement is achieved by stabilizing the value of a performance variable (such as a value of relative phase corresponding to an interlimb coordination pattern). in doing so, a subspace (i.e., manifold) is created within a state space of task - relevant elements (the df that participate in the task, for example the angular positions and velocities of two oscillating limbs), such that within the subspace the value of the performance variable remains constant. component values that do not lead to desired values of the performance variable (values outside the ucm) are restricted, whereas values that do not affect the performance variable (those within the ucm) are allowed. 2002) is using two fingers to produce a total force of, for example, 10 n. this target can be achieved by producing 5 n of force with each finger, or by pressing unequally hard with the fingers but so that the total force is 10 n (e.g., one finger produces 9 n and the other produces 1 n, or one produces 7 n while the other produces 3 n, etc.). if the force produced by each finger is plotted on orthogonal axes, the ucm is a subspace of the resulting plane, a line corresponding to forcefinger 1 + forcefinger 2 = 10 n. as long as performance falls along the ucm, the target force is achieved as one finger compensates for the other. in the ucm analysis the hypothesized stabilization of a performance variable is evaluated by computing two quantities. the first, variance along the ucm, measures the extent to which variability among the df is compensated to preserve the performance variable. variance perpendicular to the ucm measures uncompensated variability that causes the performance variable to deviate from the target. while the individual variance components are often informative, their ratio provides a compact measure of the existence and strength of a synergy. if compensated variance is greater than uncompensated variance (ratio > 1), the hypothesized performance variable is stabilized by compensation among the df the higher the ratio, the greater the amount of compensated variance, suggesting a stronger synergy (depending on the magnitude of uncompensated variance). apparent interpersonal coordination could be merely incidental (figure 2a) rather than reflecting true coordination, with the signatures of dimensional compression and reciprocal compensation, people may appear to coordinate their movements because they simultaneously execute similar motor programs, mediated by shared motor representations (garrod and pickering, 2004, 2009 ; sebanz., 2006). the studies described below contradict this alternative hypothesis and instead support the interpersonal synergy hypothesis (see figure 2b). one study applied the ucm analysis to interpersonal rhythmic movement coordination (black., 2007) and another applied principal component analysis (pca) to performance of an interpersonal precision task (ramenzoni, 2008). each actor assembles a synergy that achieves the movement pattern required by the actor 's role in the task. because actors share similar representations of the task they are able to execute similar but independent movement patterns without demonstrating the reciprocal compensation that characterizes synergies. (b) hypothetical mechanism for interpersonal coordination that involves the formation of a joint or interpersonal synergy, composed of elements of each actor 's movement system. (2007) and ramenzoni (2008) because they directly address dimensional compression and reciprocal compensation. for example, condon and ogston (1971) hand - scored videotapes of interpersonal interactions to determine when conversants spoke relative to one another. cohn and tronick (1988) evaluated when similar affect was exhibited by interacting mothers and children. (1977, 1987) quantified interpersonal coordination by hand - scoring joint - angle changes in videos of interacting participants. the degree of overall joint - angle change over time was submitted to spectral analysis to capture movement periodicity, and the spectral profiles were compared using coherence analysis to evaluate how similar two the methodology of these studies does not allow for direct evaluation of the interpersonal synergy hypothesis, although the processes of interpersonal coordination they documented is consistent with that hypothesis. programmatic studies on interpersonal rhythmic coordination (reviewed by schmidt and richardson, 2008) have also produced evidence for interpersonal synergies. coordination of rhythmically moving limbs between two individuals exhibits the same phenomena observed within individuals and predicted by the hkb model of interlimb rhythmic coordination (haken., 1985) the existence of two spontaneously stable coordination patterns (in - phase and anti - phase), transitions from anti - phase to in - phase at a critical movement frequency, critical fluctuations (increases in movement variability) preceding the transition, and a shift in the phase relation between oscillating segments when coordinating segments with different intrinsic frequencies (schmidt., 1990 ; these findings are important because according to the dynamical systems perspective, intrapersonal interlimb rhythmic coordination is a paradigmatic synergy (e.g., kugler and turvey, 1987 ; turvey, 1990 ; kelso, 1995 ; turvey and carello, 1996). this raises the possibility that interpersonal coordination likewise reflects the activity of a synergy supported by visual coupling of the actors df (schmidt and richardson, 2008 ; marsh. the first study we discuss (ramenzoni, 2008) involved pairs of participants performing an interpersonal precision task. one participant held a target (a circle), while another held a pointer through the circle without touching its sides (figure 3a). preliminary results showed that with greater task difficulty participants hand and torso movements became increasingly coordinated (as quantified by cross - recurrence quantification analysis crqa ; webber and zbilut, 1994 ; shockley., 2002 ; shockley, 2005 ; richardson., 2007). crqa quantifies the degree of shared activity between two time series by evaluating how they unfold similarly over time in a multi - dimensional (embedding) space. the proportion of body configurations shared between the time series (an overall measure of coordination) and how long they maintain similar patterns (how stable the coordination is) are among the crqa measures (see webber and zbilut, 1994). (a) depiction of the individual- (left) and interpersonal- (right) task conditions from ramenzoni (2008). (b) time series of the data projected onto the intrapersonal principle components from the individual (left) and interpersonal - task (right) conditions. the striking coordination in the interpersonal - task condition was confirmed by cross - recurrence quantification analysis, which revealed a greater degree and higher stability of coupling in that condition. displacements of participants hands, forearms, arms, and torsos were tracked in 3-d as they performed the interpersonal task or performed the subtasks (pointing, holding the target) without interacting with each other in an individual - task condition. pca identifies relations within high - dimensional datasets and maps the original data into a space whose axes (principal components) represent the dataset 's primary dimensions of variation (daffertshofer., 2004 ; those dimensions can be abstract and need not map directly onto the original dataset 's dimensions. fewer dimensions are required to account for most of the variation in the dataset (a criterion of 90% of the variance was employed in this study) than the number of original variables. vectors consisting of 12 displacement time series (4 body segments 3 spatial dimensions) were submitted to pca to identify the principal dimensions or modes of variation for each participant. for both individual- and interpersonal - task conditions six components were required to account for 90% of the variance in the original dataset a two - fold dimensional reduction. we then projected each participant 's movements onto the participant 's first principal component, creating a new variable that expressed change over time along the dimension of primary variation (figure 3b). crqa revealed a significantly greater degree of coordination as well as significantly more stable coordination between the principal components for the interpersonal- than individual - task condition. data from both participants a 24-dimensional vector (2 participants 4 body segments 3 spatial dimensions) were submitted to a second pca. for the individual - task condition six components accounted for 90% of the variance, but for the interpersonal - task condition additionally, the first component accounted for significantly more variance in the interpersonal- than the individual - task condition.. however, pca can not provide unequivocal evidence for the existence of synergies, because it does not directly measure reciprocal compensation (latash, 2008). 2007) used the ucm approach to determine whether interpersonal coordination involves dimensional compression and reciprocal compensation. they had participants oscillate a hand - held pendulum (kugler and turvey, 1987) in each hand (intrapersonal coordination) at a metronome - specified frequency to produce relative phase patterns corresponding to in - phase (0) or anti - phase (180), and had two participants, each holding a pendulum in one hand (interpersonal coordination), produce the same patterns. 2007) treated as the performance variable (with desired values of 0 or 180 for in - phase and anti - phase, respectively), and the position and velocity of each hand as component df. those variables determine each hand 's phase angle the point of the oscillating hand in its ever - repeating movement cycle and is the difference between the two hands phase angles. the ucm analysis thus quantified how variation in the position and velocity of each hand was structured (or not) to preserve the goal value. in all cases the ratio of compensated to uncompensated variance was > 1, indicating the presence of synergies for intrapersonal and interpersonal coordination. dimensional compression occurred because a lower dimensional variable,, was selectively stabilized via reciprocal compensation among the components. ratios were higher for intrapersonal (mean = 2.71) than interpersonal (mean = 1.995) coordination, paralleling findings of greater coupling strength for intrapersonal coordination (schmidt., 1998 ; the similitude between interpersonal and intrapersonal coordination was further reinforced because other manipulations, such as whether subjects were instructed to produce in - phase or anti - phase movement patterns, produced the same effects for interpersonal as for intrapersonal coordination [ratio was higher for in - phase (intrapersonal : 3.78 ; interpersonal : 2.31) than antiphase (intrapersonal : 1.64 ; interpersonal : 1.68) ]. it could be argued that the existence of stable relative phase modes already provided evidence for synergies in interpersonal rhythmic coordination, at least insofar as this evidences dimensional compression. however, an observed stable value of is mute regarding whether is selectively stabilized through synergistic compensations. no specific value of identifies the existence of a synergy, and neither a change in the mean nor the standard deviation of indicates a change in synergy strength. the black. (2007) results provide direct evidence for interpersonal synergies. they were weaker than intrapersonal synergies, but nonetheless exhibited reciprocal compensation and dimensional compression. the findings suggest synergies are not hard - wired features of an actor 's neuromuscular system, but instead they are emergent properties of perception action systems that are linked together informationally (in the case of interpersonal coordination, the informational linkage was visual). the first study we discuss (ramenzoni, 2008) involved pairs of participants performing an interpersonal precision task. one participant held a target (a circle), while another held a pointer through the circle without touching its sides (figure 3a). preliminary results showed that with greater task difficulty participants hand and torso movements became increasingly coordinated (as quantified by cross - recurrence quantification analysis crqa ; webber and zbilut, 1994 ; shockley., 2002 ; shockley, 2005 ; richardson., 2007). crqa quantifies the degree of shared activity between two time series by evaluating how they unfold similarly over time in a multi - dimensional (embedding) space. the proportion of body configurations shared between the time series (an overall measure of coordination) and how long they maintain similar patterns (how stable the coordination is) are among the crqa measures (see webber and zbilut, 1994). (a) depiction of the individual- (left) and interpersonal- (right) task conditions from ramenzoni (2008). (b) time series of the data projected onto the intrapersonal principle components from the individual (left) and interpersonal - task (right) conditions. the striking coordination in the interpersonal - task condition was confirmed by cross - recurrence quantification analysis, which revealed a greater degree and higher stability of coupling in that condition. displacements of participants hands, forearms, arms, and torsos were tracked in 3-d as they performed the interpersonal task or performed the subtasks (pointing, holding the target) without interacting with each other in an individual - task condition. pca identifies relations within high - dimensional datasets and maps the original data into a space whose axes (principal components) represent the dataset 's primary dimensions of variation (daffertshofer., 2004 ; forner - cordero., those dimensions can be abstract and need not map directly onto the original dataset 's dimensions. fewer dimensions are required to account for most of the variation in the dataset (a criterion of 90% of the variance was employed in this study) than the number of original variables. vectors consisting of 12 displacement time series (4 body segments 3 spatial dimensions) were submitted to pca to identify the principal dimensions or modes of variation for each participant. for both individual- and interpersonal - task conditions six components were required to account for 90% of the variance in the original dataset a two - fold dimensional reduction. we then projected each participant 's movements onto the participant 's first principal component, creating a new variable that expressed change over time along the dimension of primary variation (figure 3b). crqa revealed a significantly greater degree of coordination as well as significantly more stable coordination between the principal components for the interpersonal- than individual - task condition. data from both participants a 24-dimensional vector (2 participants 4 body segments 3 spatial dimensions) were submitted to a second pca. for the individual - task condition six components accounted for 90% of the variance, but for the interpersonal - task condition there was greater dimensional reduction and just four components were required. additionally, the first component accounted for significantly more variance in the interpersonal- than the individual - task condition. however, pca can not provide unequivocal evidence for the existence of synergies, because it does not directly measure reciprocal compensation (latash, 2008). black. (2007) used the ucm approach to determine whether interpersonal coordination involves dimensional compression and reciprocal compensation. they had participants oscillate a hand - held pendulum (kugler and turvey, 1987) in each hand (intrapersonal coordination) at a metronome - specified frequency to produce relative phase patterns corresponding to in - phase (0) or anti - phase (180), and had two participants, each holding a pendulum in one hand (interpersonal coordination), produce the same patterns. black. (2007) treated as the performance variable (with desired values of 0 or 180 for in - phase and anti - phase, respectively), and the position and velocity of each hand as component df. those variables determine each hand 's phase angle the point of the oscillating hand in its ever - repeating movement cycle and is the difference between the two hands phase angles. the ucm analysis thus quantified how variation in the position and velocity of each hand was structured (or not) to preserve the goal value. in all cases the ratio of compensated to uncompensated variance was > 1, indicating the presence of synergies for intrapersonal and interpersonal coordination. dimensional compression occurred because a lower dimensional variable,, was selectively stabilized via reciprocal compensation among the components. ratios were higher for intrapersonal (mean = 2.71) than interpersonal (mean = 1.995) coordination, paralleling findings of greater coupling strength for intrapersonal coordination (schmidt., 1998 ; the similitude between interpersonal and intrapersonal coordination was further reinforced because other manipulations, such as whether subjects were instructed to produce in - phase or anti - phase movement patterns, produced the same effects for interpersonal as for intrapersonal coordination [ratio was higher for in - phase (intrapersonal : 3.78 ; interpersonal : 2.31) than antiphase (intrapersonal : 1.64 ; interpersonal : 1.68) ]. it could be argued that the existence of stable relative phase modes already provided evidence for synergies in interpersonal rhythmic coordination, at least insofar as this evidences dimensional compression. however, an observed stable value of is mute regarding whether is selectively stabilized through synergistic compensations. no specific value of identifies the existence of a synergy, and neither a change in the mean nor the standard deviation of indicates a change in synergy strength. the black. (2007) results provide direct evidence for interpersonal synergies. they were weaker than intrapersonal synergies, but nonetheless exhibited reciprocal compensation and dimensional compression. the findings suggest synergies are not hard - wired features of an actor 's neuromuscular system, but instead they are emergent properties of perception action systems that are linked together informationally (in the case of interpersonal coordination, the informational linkage was visual). the evidence reviewed here is consistent with claims that movement system df residing in different actors are coupled to form low - dimensional, reciprocally compensating synergies. this may be controversial, because interpersonal synergies span organisms and extend beyond boundaries of skin, with the coupling among movement system df achieved (in these studies) visually. might reside beyond the boundaries of an organism, or more specifically beyond a particular, singular component of an individual 's brain. the control evident during interpersonal coordination may be an emergent property of a social perception, 2008a, 2010 ; schmidt and richardson, 2008 ; marsh., 2009 ; shockley., 2009). a key point to consider with regard to situating this approach scientifically and philosophically this echoes the distinction between interaction - dominant dynamics and component - dominant dynamics described by van orden. component - dominant dynamics is assumed by classical cognitivism, with its emphasis on static modules that perform their functions in relative isolation of the activity of other modules. interaction - dominant dynamics emphasizes interactions between components rather than their structure, entailing a functional, dynamical approach to understanding coordinated action. instead of focusing on component structures, this question raises another key point : the interpersonal synergy approach, with its roots in self - organizing complex systems, entails notions of mechanism and causality that are broader than the usual (newtonian) sense of the terms as involving only efficient causes, the kinds of forceful interactions produced by colliding cogs or billiard balls (rosen, 1991 ; juarrero, 1999 ; van orden., 2005). complex systems exhibit circular causality ; bottom - up processes give rise to macroscopic patterns that simultaneously constrain the components from the top down. constraints play the role of causal mechanisms in complex systems insomuch as constraints allow or deny certain states. constraints limit the df of a system, but do not cause the system to take on particular states by virtue of forceful pushes (local, efficient causes). control (manipulation of the movement system) first entails coordination (organization of the movement system), as anticipated by bernstein (1967). control parameters are constraints that guide the movement system through sequences of stable states (identified by values of order parameters). in rhythmic movement coordination, is the order parameter and movement frequency is a control parameter that, when varied, results in transitions between coordination patterns. movement frequency has been identified as a control parameter for interpersonal rhythmic coordination (schmidt., 1990), but the identification of control parameters for other interpersonal synergies remains an important avenue for future research. possibilities include attention to movement information (richardson., 2007 ; schmidt., 2007), individual or social intentions or goals, perceived social differences, or rapport and liking (e.g., chartrand and bargh, 1999 ; hove and risen, 2009 ; marsh., 2009 ; miles., 2010), to name a few. the alternative hypothesis to interpersonal synergies is that coordination observed between agents results because they execute similar but independent motor programs anchored in shared representations neurons in the pre - motor cortex that are activated when observing others performing actions suggest a common neural basis for motor control and action understanding. watching someone engage in an action is hypothesized to activate the actor 's own motor representation of that action, resulting in a shared motor representations. it is claimed that shared representations facilitate understanding others actions (e.g., blakemore and decety, 2001 ; richardson and dale, 2005 ; sebanz., 2006), which may explain why motor coordination occurs spontaneously in social contexts. this would not predict the synergistic nature of interpersonal coordination that we have described, however. although this approach might predict dimensional compression (pca would be sensitive to any correlations between the two actors movements), it would not predict reciprocal compensation. reciprocal compensation means that the actors movements are not independent, directly contradicting this hypothesis. interpersonal coordination is a fundamental means of forming social units (marsh., 2009)., 1996 ; chartrand and bargh, 1999 ; hove and risen, 2009 ; marsh., 2009). being psychologically distanced from another individual can inhibit the emergence of interpersonal synergies (miles., 2010). interpersonal synergies facilitate memory for those with whom we interact (miles., 2010) and can more generally facilitate performance of social cognitive or linguistic tasks (richardson and dale, 2005 ; shockley., 2009). synergies might also be a mechanism for interpersonal coordination in team sports (e.g., passos., 2009). for example, highly effective teams might exhibit a greater degree of reciprocal compensation than poorly effective ones. developing a detailed understanding of interpersonal synergies may have broad and significant implications for understanding social perception the interpersonal synergy hypothesis can be subjected to additional tests, using a range of paradigms and complementary methods such as the ucm analysis and pca. if the hypothesis is supported movement science will be charged with the important task of broadening its conceptions of fundamental processes of coordination and control, and areas such as social psychology or sports psychology might benefit from the development of new tools and paradigms. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.	we present the perspective that interpersonal movement coordination results from establishing interpersonal synergies. interpersonal synergies are higher - order control systems formed by coupling movement system degrees of freedom of two (or more) actors. characteristic features of synergies identified in studies of intrapersonal coordination dimensional compression and reciprocal compensation are revealed in studies of interpersonal coordination that applied the uncontrolled manifold approach and principal component analysis to interpersonal movement tasks. broader implications of the interpersonal synergy approach for movement science include an expanded notion of mechanism and an emphasis on interaction - dominant dynamics.
nephritis is one of the most severe complications of systematic lupus erythematosus (sle).1 up to 60% of adults and 80% of children with sle develop lupus nephritis (ln).2 the clinical presentation of kidney involvement is highly variable, ranging from mild asymptomatic proteinuria to rapidly progressive glomerulonephritis. biological features generally include varying degrees of glomerular involvement with proteinuria that is nephrotic in up to 65% of cases, as well as hematuria with red - cell casts and/or acute renal failure.1 diffuse proliferative ln is the most common histological variant. furthermore, it has the worst prognosis, with a reported 17% 5-year survival without treatment.3 progress in immunosuppressive therapy has improved renal and global survival. five - year patient survival was approximately 55% in 1970 versus up to 80% a decade later,4 and currently the 5-year survival rate reaches about 90%.3 prognostic factors of renal involvement include demographic (age, sex, race), genetic and immunological features (anti double - stranded dna [dsdna ], anti - c1q, antiphospholipid antibodies), histopathological findings, clinical and laboratory signs (high serum creatinine, nephrotic syndrome, hypertension), and are affected also by treatment regimens and patient compliance.5 in morocco, no data describing ln have been published previously. in order to make a clear picture, we reviewed retrospectively 114 cases of ln followed in our unit between 2001 and 2010. the aim of our study was to analyze the clinical and histological features of patients with ln and to evaluate the outcome of ln with special regards to proliferative and membranous forms. between january 2001 and december 2010, 114 cases of ln were diagnosed treated and followed up by the department of nephrology, dialysis, and renal transplantation, ibn sina university hospital, rabat, morocco. all patients included in this single - center retrospective analysis fulfilled at least four of the revised american rheumatism association criteria for sle.6 ln was defined as positive proteinuria (persistent proteinuria of greater than 0.5 g per day) and/or active urinary sediment (hematuria) and/or presence of renal failure (increase in serum creatinine of more than 1.5 mg / dl). histological findings were classified according to the international society of nephrology / renal pathology society 2003 classification of lupus nephritis.7 all biopsies performed before 2004 were reviewed by our pathologist and reclassified according to the new classification. analyzed parameters included : (1) demographic data age, sex ; (2) clinical signs blood pressure and american rheumatism association criteria ; (3) biological parameters serum creatinine, urinalysis (proteinuria and hematuria), antibodies against dsdna, antiphospholipid antibody (lupus anticoagulant, anticardiolipin antibody), complement components c3 and c4 ; (4) immunosuppressive therapy ; and (5) outcomes remission, relapse, end - stage renal disease (esrd), and death. hypertension was defined as blood pressure superior to 140/90 mmhg or the use of any antihypertensive medications. we adopted outcome criteria for ln as defined by boumpas:8 complete remission was defined as stabilization or improvement in renal function, resolution of urine - sediment abnormalities (absence of hematuria or cellular cast), proteinuria 2 g / day or doubling if > 3.5 g / day after response and/or activity increase in urine sediment and/or increase in serum creatinine. all patients with proliferative classes as well as class v ln received a 3-day pulse of methylprednisolone, followed by oral prednisone (1 mg / kg / day). prednisone was tapered progressively in order to reach 10 mg / day 6 months after the beginning of the treatment. immunosuppressive treatment consisted of six monthly pulses of intravenous (iv) cyclophosphamide (cyc ; 5001000 mg / month) as induction of renal remission. maintenance therapy consisted of six trimonthly pulses of iv cyc or oral mycophenolate mofetil (mmf ; 23 g / day) or oral azathioprine (aza) 100 mg / day for 2 years, as well as oral corticosteroid (cs) therapy. the patients were seen monthly with a physical and biological assessment prior to cyc pulse. during and after maintenance treatment, patients were seen each trimester. patients presenting with a complication during follow - up were managed in the emergency room of our unit. the statistical package spss 17.0 (ibm, armonk, ny, usa) was used to analyze sample data. there were 101 females (88.6%) and 13 males (11.4%), making a sex ratio of 7.76:1. initial presentation of sle in the present work was mostly with joint and skin manifestations in 85% and 74%, respectively. time between diagnosis of sle and the onset of renal disease was 9.7 3.6 months. at admission, 75.4% of the patients presented with ln at the diagnosis of lupus. also, all the reported initial ln flares in this study were the first ones that occurred in our patients. moreover, at presentation, 38 (33.3%) of our patients had hypertension. nephrotic syndrome was found in 60 patients (52.6%) and renal failure in 68 patients (59.6%). low components of complement c3 and c4 were seen in 71.9% and 65.5%, respectively. of 96 patients in whom renal biopsy was performed, three (3.1%) had ln class i, two (2%) class ii, ten (10.4%) class iii, sixty (62.5%) class iv, eight (8.3%) class v, and three (3.1%) class vi. ten (10.4%) patients had combined classes (eight patients had class v combined with class iv, and two others had class v combined with class iii). it is important to note that all proliferative ln classes showed signs of activity in our series. two patients were lost to view before initial immunosuppressive therapy ; therefore, they were not included in further analysis. forty - two of them (82.35%) relapsed at least once (figure 1). at 6 months, of all patients, 24 (21.42%) progressed to esrd, and 18 (16.07%) died because of infectious complications in ten patients, severe neurologic state in four and acute cardiovascular events in four patients (table 2). class iii occurred in ten patients, class iv in 60 patients, and combined classes in ten (10.4%) patients. nephrotic syndrome was present in 61 (76.2%) patients, active urinary sediment in 63 (78.7%), renal failure in 55 (68.7%) with mean serum creatinine of 4.46 mg / dl, and hypertension in 35 (43.7%). all patients with proliferative classes received a 3-day pulse of methylprednisolone followed by oral prednisone (1 mg / kg / day) and six monthly pulses of iv cyc (5001000 mg / month) as induction of renal remission. maintenance therapy was six trimonthly pulses of iv cyc in 55 patients (68.75%), 2 years of 100 mg / day aza in 17 patients (21.25%) or 2 g / day of mmf in two patients (2,5%), as well as cs therapy. complete and partial remission were observed in 21 patients (26.25%) and eleven patients (13.75%), respectively. forty - seven of our ln active proliferative class patients (58.75%) relapsed. at the end of the follow - up, the total number of patients who progressed to esrd was 19 (23.75%). during the follow - up, 11 (13.75%) eight (7%) patients with mean age of 28.1 10 (1443) years had membranous ln. at the time of biopsy, six (75%) of them had nephrotic syndrome with mean proteinuria of 3.8 2.24 g / day. only three (37.5%) had hematuria, while hypertension was seen in one (12.5%) patient. renal failure was present in three (37.5%) patients, with mean serum creatinine of 1.68 1.35 mg / dl. another feature of class v was a high frequency of negative anti - dsdna antibodies (75%). for induction of renal remission, all patients except one received pulses of methylprednisolone followed by oral prednisone at 1 mg / kg / day on a regressive regimen. four patients had six monthly pulses of iv cyc (5001000 mg / month). as maintenance therapy, three of them had six trimonthly pulses of iv cyc and one patient had aza 100 mg / day. one patient developed esrd and another died because of a neurologic complication. in our study, global survival was 84.5% at 1 year and 76% at 5 years (figures 3 and 4). among the clinical features at presentation that were associated with occurrence of esrd or death were short mean time between sle and ln (p = 0.04), renal failure at admission (p = 0.01), and anemia (p = 0.03) (table 3). regarding our patients treatment, induction therapy was based on cyc in all cases. for maintenance treatment, most of our patients received trimonthly pulses of cyc, while aza and mmf were used in 13.75% and 25%, respectively. class iii occurred in ten patients, class iv in 60 patients, and combined classes in ten (10.4%) patients. nephrotic syndrome was present in 61 (76.2%) patients, active urinary sediment in 63 (78.7%), renal failure in 55 (68.7%) with mean serum creatinine of 4.46 mg / dl, and hypertension in 35 (43.7%). all patients with proliferative classes received a 3-day pulse of methylprednisolone followed by oral prednisone (1 mg / kg / day) and six monthly pulses of iv cyc (5001000 mg / month) as induction of renal remission. maintenance therapy was six trimonthly pulses of iv cyc in 55 patients (68.75%), 2 years of 100 mg / day aza in 17 patients (21.25%) or 2 g / day of mmf in two patients (2,5%), as well as cs therapy. complete and partial remission were observed in 21 patients (26.25%) and eleven patients (13.75%), respectively. forty - seven of our ln active proliferative class patients (58.75%) relapsed. at the end of the follow - up, the total number of patients who progressed to esrd was 19 (23.75%). during the follow - up, 11 (13.75%) patients died. eight (7%) patients with mean age of 28.1 10 (1443) years had membranous ln. at the time of biopsy, six (75%) of them had nephrotic syndrome with mean proteinuria of 3.8 2.24 g / day. only three (37.5%) had hematuria, while hypertension was seen in one (12.5%) patient. renal failure was present in three (37.5%) patients, with mean serum creatinine of 1.68 1.35 mg / dl. another feature of class v was a high frequency of negative anti - dsdna antibodies (75%). for induction of renal remission, all patients except one received pulses of methylprednisolone followed by oral prednisone at 1 mg / kg / day on a regressive regimen. four patients had six monthly pulses of iv cyc (5001000 mg / month). as maintenance therapy, three of them had six trimonthly pulses of iv cyc and one patient had aza 100 mg / day. one patient developed esrd and another died because of a neurologic complication. in our study, global survival was 84.5% at 1 year and 76% at 5 years (figures 3 and 4). among the clinical features at presentation that were associated with occurrence of esrd or death were short mean time between sle and ln (p = 0.04), renal failure at admission (p = 0.01), and anemia (p = 0.03) (table 3). regarding our patients treatment, induction therapy was based on cyc in all cases. for maintenance treatment, most of our patients received trimonthly pulses of cyc, while aza and mmf were used in 13.75% and 25%, respectively. it was performed on a cohort of 114 patients followed up in a single center. the moroccan population has arisen from a great intermingling of different populations throughout history, mainly composed of caucasians (berbers and arabs) and black africans. in fact, according to the european journal of human genetics, moroccans from northwestern africa are genetically closer to iberians and other south europeans than to middle easterners or sub - saharan africans.9 ln has been described in various ethnic groups, including caucasians, asians, latinos, and black africans,10 but little literature exists on ln features in north african patients, in particular from morocco. our goal for this retrospective study was to examine ln features among a moroccan population that is large and ethnically heterogeneous. systemic manifestations in this work did not differ from what was observed in other studies, with predominance of joint and skin manifestations in 85% and 74%, respectively. renal manifestations in this study were marked by a high frequency of renal failure, nephrotic syndrome, hematuria, and hypertension. similar results were seen in most studies,1114 especially in the tunisian cohort of beji (table 4). this interval is very short compared to the 3 years observed in the study of mok,15 between 3 and 4.4 years in the study of brugos,16 and 4 years in the study of chrysochou.11 most studies with ln were performed according to the 1995 world health organization classification. in these studies,13,1626 proliferative classes (particularly class iv) were predominant, occurring in up to 70% of patients. our work was based on the 2003 international society of nephrology / renal pathology society classification of ln, but our findings are consistent with what was observed in previous studies. second, we described a higher frequency of membranous glomerulonephritis similar to that published when combined classes are included, except in the senegalese study,23 where class v was the most dominant form of ln, observed in more than half of patients. brugos reported a similar result with a percentage of 17.9% (table 5). combination cs and cyc administered either traditionally (monthly iv cyc) or in a modified regimen (smaller doses of cyc given at fortnightly intervals over a shortened treatment duration (euro - lupus) can induce a complete or partial remission in more than 80% of patients with proliferative lupus nephritis (pln).27 aza and mmf emerged recently as a good alternative to cyc, especially as maintenance treatment.27 however, remission was recently reported to vary from 30% to 81%.28 also, the incidence of ln flares varies in different studies between 27% and 66%.8 the discrepancies obtained from various studies can be attributed easily to (1) a difference in definitions, (2) racial and environmental differences between the groups of patients studied, and (3) disparities on treatment regimens. in our study, most patients with pln had iv cyc administered monthly as induction and trimonthly as maintenance treatment because of efficacy and low cost. remission occurred in less than half of cases, 60% of them relapsed at least once, 21% of patients progressed to esrd, and 16% died. similar results were reported by beji :13 of the 211 ln cases followed up for 103 months, remission was obtained in 55.3%, relapses occurred in 51% of cases, 14.7% progressed to esrd, and death was noted in 16.63%. in our study, it appears clear that ln in our population was particularly severe, compared with other published data.29 a number of factors may account for such a poor prognosis. most lupus nephritis patients presented at our nephrology unit with severe clinical and biological features. at admission, 38 (33.3%) patients had hypertension. nephrotic syndrome was found in 60 patients (52.6%) and renal failure in 68 patients (59.6%). furthermore, renal biopsy at admission showed mostly proliferative lupus classes : 10.4% class iii, 62.5% class iv, and 10.4% of patients had combined classes (iii + v and iv + v). the severity of this disease in our patients can not be related to race or ethnicity, since it has been recently established that moroccans are genetically closer to iberians and other southern europeans than to middle easterners or sub - saharan africans.9 besides, ln in southern europeans is less severe than that found in our series.29 in fact, low remissions and high relapses in our context are mainly due to irregular follow - up, not allowing for correct surveillance in most cases. the low economic background of 57% of our patients probably plays a determining role in this condition, as most patients came from regions of the country in which there are no nephrologists in the local hospitals. thus, they were referred to our university hospital for specialized care. in many cases, poverty and distance discourage patients from attending their consultation appointments. eighty percent of patients were lost to view at 5 years, reflecting the short follow - up period, which was a major limitation in our study. the economic and socioeconomic background as well as the poor level of education encountered in most ln patients in our series might affect patients adherence to treatment, though this issue was nt evaluated in this study. nonetheless, low compliance with prescribed treatments is highly suspected by medical staff and seldom admitted by patients. on the other hand, the high rate of relapse in our series may be explained by the maintenance - treatment regimen, mostly based on trimonthly 2-year cyc pulses. the main cause of death in ln patients is overtreatment, which may lead to life - threatening morbidity principally represented by severe infection.29 in our study, death was mostly due to infectious complications. in our opinion, improvement in ln outcome in our country can not be achieved without a thorough improvement of our health system. bringing health structures closer to patients and training more doctors, especially nephrologists, throughout the country would help in diagnosing lupus at an early stage and perhaps help in preventing the development of some of its most devastating complications, such as nephritis. besides, the generalization of health insurance among the moroccan population can also play an undeniable role in allowing treatment continuance, especially in low - income patients. furthermore, the use of mmf or aza as a maintenance therapy should be used on a larger scale in our patients, since these drugs are currently recommended.30 moreover, a longer maintenance - therapy period would also help in preventing relapses, although to date there is still no available adequate data regarding when maintenance treatment can be withdrawn.30 symptoms, histological findings, and outcomes were similar to those of non - caucasian series. our results revealed severe ln with predominance of proliferative forms and severe renal manifestations with poor renal and overall survival.	backgroundthere is wide variation in clinical presentation and outcome of lupus nephritis (ln) among different ethnic groups. few data for ln exist on north africans, especially those from morocco. the aim of our study was to review retrospectively the features and outcome of ln in moroccan patients.patients and methodswe performed a single - center retrospective study. a total of 114 patients with ln were included. all patients met american rheumatism association criteria. ln was classified according to the international society of nephrology / renal pathology society classification. we adopted previously defined outcome criteria for ln.resultsthere were 101 females and 13 males, with a mean age of 29.9 years. at first presentation, we noted hypertension in 33%, hematuria in 76%, nephrotic syndrome in 53%, and renal failure in 60% of cases. renal biopsy revealed predominant proliferative classes in more than 80% of patients. patients received different regimens mainly based on intravenous cyclophosphamide. after a mean follow - up of 22 months, remission occurred in 45.5%, relapses in 82%, end - stage renal failure in 21%, and death in 16% of cases. infection and neurological and cardiovascular diseases were the most frequent causes of death.conclusionln seems to be severe in our study, with a predominance of proliferative forms, severe renal manifestations, and poor renal and overall survival.
the following online material is available for this article : expression abundance of 69 dna repair genes during rat liver regeneration. comparison of relative mrna levels in regenerating liver detected by real - time rt - pcr and affy - metrix rat genome 230 2.0 microarray.	rapidly proliferating tissue may require enhanced dna repair capacity in order to avoid fixation of promutagenic dna lesions to mutations. partial hepatectomy (ph) triggers cell proliferation during liver regeneration (lr). however, little is known on how dna repair genes change and how they are regulated at the transcriptional level during lr. in the present study, the rat genome 230 2.0 array was used to detect the expression profiles of dna repair genes during lr, and differential expression of selected genes was confirmed by real - time rt - pcr. 69 dna repair genes were found to be associated with lr, more than half of which distributed in a cluster characterized by a gradual increase at 2472h and then returning to normal. the expression of base excision repair- and transcription - coupled repair - related genes was enhanced in the early and intermediate phases of lr, whereas the expression of genes related to hr, nhej and dna cross - link repair, as well as dna polymerases and related accessory factors, and editing or processing nucleases, were mainly enhanced in the intermediate phase. the expression changes of genes in dna damage response were complicated throughout the whole lr. our data also suggest that the expression of most dna repair genes may be regulated by the cell cycle during lr.
the typical patient presenting with myxedema coma is a woman in her 70s and the cardinal manifestation is deterioration of mental status. physical findings often include hypothermia without shivering, bradycardia, hypoventilation, macroglossia, hoarseness, dry skin, and delayed tendon reflexes ; if hypothyroidism is long standing, there will also be accumulation of fluid rich in mucopolysaccharides in extracellular space manifesting as peripheral nonpitting edema, ascites, pleural and pericardial effusion. routine laboratory evaluation may indicate anemia, hyponatremia, hypercholesterolemia, increased serum lactate dehydrogenase and creatine kinase. typical electrocardiogram findings are varying degrees of blocks, low voltage, prolonged qt interval, and flattened or inverted t waves. finally, the eeg may show a pattern typical to metabolic coma, including triphasic waves.1 hepatic encephalopathy is a metabolic coma, the hallmark of which is hyperammonemia. although not all patients with hepatic encephalopathy present with hyperammonemia, and hyperammonemia is not pathognomonic for hepatic encephalopathy, the common practice is to consider patients with a liver disease, hyperammonemia, and coma as suffering from hepatic encephalopathy. the differentiation between myxedema coma and hepatic encephalopathy, conditions which share many similarities, is of utmost importance and carries therapeutic implication. an 82-year - old woman was admitted to the hospital in a state of coma. she had been alert and functioning until 1 week before admission, when she gradually became lethargic and unresponsive. the patient had a history of hypothyroidism, vitiligo, essential hypertension, depressive disorder, and mild impairment of liver function, which had not been investigated in the past. her chronic medication included amlodipine, sertraline, furosemide, ramipril, atenolol, and levothyroxine. on physical examination she was in a state of coma with a glasgow coma score of 5. her rectal temperature was 36.1c, heart rate was 56 beats per minute, blood pressure 118/70 mmhg, and respiration rate 20 per minute. neurologically, she was unresponsive, achilles tendon reflexes were absent bilaterally, and flapping tremor was not elicited. blood tests revealed macrocytosis of 110 m (78102 m), severe vitamin b12 deficiency 74 pg / ml (200800 pg / ml), and no antiparietal cell antibodies. u / l), and mild abnormalities in enzymatic, cholestatic, and biosynthetic liver function tests were noted : aspartate aminotransferase (ast) 232 u / l (035 u / l), alanine aminotransferase (alt) 65 u / l (035 u / l), gamma glutamyl transferase (ggt) 69 u / l (194 u / l), alkaline phosphatase (alp) 142 u / l (30120 u / l), bilirubin total 0.9 mg / dl (0.31 mg / dl), international normalized ration (inr) 1.44, albumin 2.4 g / dl (3.55.5 g / dl). her serum ammonia level was distinctly elevated, 194 mol / l (647 mol / l). hepatic veins were normal in diameter and no hepatofugal flow in the portal vein was detected. a liver scan demonstrated colloid shift to the spleen and bone marrow, consistent with chronic liver disease. further evaluation of liver disease revealed negative serology for hiv, hepatitis b and c viruses, coxiella burnetii phase 1 and 2, antismooth muscle, anti - lkm, antisoluble liver antigen and antimitochondrial antibodies. an elevated antinuclear factor titer, 1:640 (negative at 1:40) and hypergammaglobolinemia, igg concentration of 3,200 mg / dl (6141,295 mg / dl) were suggestive of autoimmune hepatitis type 1. at this point a drop in blood pressure was noted and was evaluated by a 1-mcg acth test, which revealed adrenal insufficiency : cortisol 0 minute150 nmol / liter (171536 nmol / l at 8:00 a.m.) and cortisol 30 minutes200 nmol / l (> 550 nmol / l or an increase of > 200 nmol / l). treatment of hepatic encephalopathy and adrenal insufficiency with lactulose, neomycin, and hydrocortisone was initiated. blood pressure normalized ; however, only a mild improvement in the state of consciousness was noticed ; liver function test results worsened and her ammonia level did not change. thyroid function test results, received 3 days after admission, revealed severe hypothyroidism (thyroid stimulating hormone 49 u / ml [0.54.7 u / ml ] and free t4 550 nmol / l or an increase of > 200 nmol / l). treatment of hepatic encephalopathy and adrenal insufficiency with lactulose, neomycin, and hydrocortisone was initiated. blood pressure normalized ; however, only a mild improvement in the state of consciousness was noticed ; liver function test results worsened and her ammonia level did not change. thyroid function test results, received 3 days after admission, revealed severe hypothyroidism (thyroid stimulating hormone 49 u / ml [0.54.7 u / ml ] and free t4 < 2.57 ultrasound examination displayed an enlarged thyroid gland with multiple ill - defined nodules characteristic of hashimoto s thyroiditis. treatment with intravenous levothyroxine 100 g was commenced and resulted in a rapid (within 24 hours) regain of consciousness and a drop in serum ammonia level to 64 g / dl. soon thereafter the eeg pattern normalized, ck and liver function test results returned to baseline. after regaining full consciousness, the patient reported that she had discontinued levothyroxine therapy 4 months before admission. we present a patient with hypothyroidism, adrenal insufficiency, suspected autoimmune hepatitis and atrophic gastritis a combination that is consistent with autoimmune polyglandular syndrome type 2. discontinuation of levothyroxine therapy resulted in hyperammonemic coma with severe hypothyroidism that was unresponsive to lactulose and neomycin, but which was resolved after thyroid hormone replacement. in this patient hyperammonemic coma an alternative explanation may be that hypothyroidism was a precipitant for decompensation of liver disease and thus hyperammonemic coma was the manifestation of hepatic encephalopathy. myxedema coma and hepatic encephalopathy share many clinical features : coma, ascites, pleural effusion, peripheral edema, clubbing, anemia, hypercholesterolemia, hyponatremia, hypoglycemia, elevated liver enzymes level including lactate dehydrogenase and metabolic pattern on eeg. an eeg pattern of triphasic waves, elevated liver enzymes, and hyperammonemia are rare and unfamiliar features of myxedema coma, which may divert the clinical judgment from the correct diagnosis and be confused with hepatic encephalopathy. an eeg pattern of triphasic waves was first described in hepatic encephalopathy by bickford and butt in 1955 and became synonymous with hepatic encephalopthy (he).2 nevertheless, triphasic waves are not pathognomonic for hepatic encephalopathy and have been reported in other metabolic encephalopathies, the most common of which are renal failure and anoxic injury.3 to the best of our knowledge, only 1 report in the literature describes triphasic waves during hypothyroidism.4 elevated liver enzymes may reflect the effect of hypothyroidism on the liver or be related to a concomitant liver disease. the association of hypothyroidism and liver diseases including autoimmune hepatitis, primary biliary cirrhosis, and chronic hepatitis c is not uncommon (613%).57 the liver is not considered a hormonally regulated organ. an increase in liver enzymes concentration, ast more than alt, is often found.8 an animal study in hypothyroid rats has, however, shown that, whereas alt function (production of pyruvate) increases, ast function (production of oxaloacetate) decreases.9 microscopic changes including central congestive fibrosis have been described in myxedema ascites.10 in our patient, elevated liver enzymes, ast more than alt, may have been a consequence of either hypothyroidism or autoimmune hepatitis. a high titer of antinuclear antibody (ana) and hypergammaglobolinemia in the setting of autoimmune polyglandular syndrome type 2 are suggestive of autoimmune hepatitis, yet a definite diagnosis was not reached, as the patient declined a liver biopsy. a retrospective review of the medical history of the patient confirmed that increased plasma levels of liver enzymes in the presence of normal tsh had repeatedly been recorded several years before admission. hence, in the presence of euthyroidism, it is reasonable that some other smoldering chronic liver disease, most probably autoimmune hepatitis, was present before the occurrence of hypothyroidism. rare causes of hyperammonemia include drug toxicity (e.g., valproic acid, salicylates, and 5 fluorouracil), and urinary tract infection with a urease - producing organism, such as klebsiella pneumonia, proteus mirabilis, corynebacterium species, or staphylococcus species.1113 hyperammonemia in our patient was at first attributed by us to decompensation of a chronic liver disease. evaluation of our patient identified constipation and exacerbation of autoimmune hepatitis to be possible inciting events for hyperammonemia. however, lack of response to lactulose, neomycin and steroids did not support this assumption. prompt response to thyroid hormone replacement suggested that hyperammonemia was either an atypical manifestation of severe hypothyroidism or that hypothyroidism was the inciting event for liver decompensation and hepatic encephalopathy in the setting of a chronic liver disease. to date, 4 cases of hyperammonemia and myxedema coma have been described in the literature.1417 all reported cases, including ours, describe patients with an undiagnosed or fully compensated liver disease. all cases presented a clinical picture of hyperammonemic coma, unresponsive to lactulose and neomycin therapy, which improved only after thyroid hormone replacement. hyperammonemia has also been described in 3 patients with hypothyroid myopathy and no known previous liver disease, in whom a concomitant increase in transaminases, and hyperammonemia attributed to increased catabolism of muscle, completely resolved after gaining euthyroidism. hyperammonemia in this setting did not result in any neurological impairment.18 hyperammonemia in hypothyroidism may be explained by pathophysiological studies of the urea cycle in this condition. in a rat liver model, hypothyroidism was associated with an increased urea synthesis, attributed to an increase in urea cycle enzyme activity.9,19 however, when studied in hypothyroid women, a decreased urea synthesis rate, in comparison with values measured in euthyroidism, was found.20 inefficient urea synthesis during hypothyroidism, as was demonstrated in vivo, is expected to result in an increased ammonia level. moreover, an increased load of nitrogen products during hypothyroidisim may aggravate the hyperammonemic state. several mechanisms can explain the suspected increased nitrogen load : 1) decreased protein synthesis and increased protein catabolism (attributed to a decrease in growth hormone and to hypothyroid myopathy)21,22 ; 2) decreased intestinal motility that promotes bacterial production of ammonia and augments its absorption ; and 3) decreased glutamine synthetase activity that may diminish utilization of glutamine by the urea cycle in the liver.9 despite the available information on mechanisms by which hypothyroidism may impair liver functions, the exact role of hypothyroidism in hyperammonemic coma as a direct inciting event or a precipitant for decompensation of liver disease has not yet been resolved. it is of utmost importance to bear in mind the possibility of combined hypothyroidism and liver disease in hyperammonemic coma and to carefully search for clues to each of these conditions in physical examination, laboratory results, and imaging studies. a careful physical examination may reveal findings typical of myxedema such as dry cool skin, coarse, sparse hair, and macroglossia, or findings suggestive of cirrhosis : jaundice, spider angiomas, caput medusa, dupuytren s contracture, palmar erythema, fetor hepaticus, gynecomastia, hair loss, paper money skin, parotid enlargement, clubbing, and terry s nails (white nails), all of which were missing in our patient. peripheral edema is typically nonpitting in myxedema, in contrast to the typical pitting edema in liver disease. tendon reflexes are delayed in myxedema coma as was noticed in our patient, whereas hyperreflexia, asterixis, and flapping tremor are characteristic of hepatic encephalopathy. laboratory work - up may reveal pancytopenia caused by hypersplenism, a characteristic of cirrhosis or elevated cpk and macrocitic anemia more typical to myxedema. finally, imaging studies may help demonstrate pericardial effusion or an enlarged thyroid gland, characteristic of myxedema or a nonhomogenous liver with hepatofugal flow in hepatic veins, characteristic of cirrhosis. in conclusion : in patients with an elevated ammonia level, altered mental status, and liver disease, who do not have a clear inciting event for decompensation of liver disease, overwhelming evidence of hepatic decompensation, or who do not respond to appropriate therapy for hepatic encephalopathy, hypothyroidism should be considered and evaluated. increased nitrogen load : excess of dietary proteinazotemiaconstipationelectrolytes and metabolic imbalance : hypokalemiaalkalosishypoxiahyponatremiahypovolemiadrugs : narcotics, tranquilizers, sedatives, diureticsmiscellaneous : infectionsurgerysuperimposed acute liver diseaseprogressive liver diseaseportal systemic shunt increased nitrogen load : excess of dietary proteinazotemiaconstipation excess of dietary protein electrolytes and metabolic imbalance : hypokalemiaalkalosishypoxiahyponatremiahypovolemia narcotics, tranquilizers, sedatives, diuretics narcotics, tranquilizers, sedatives, diuretics infectionsurgerysuperimposed acute liver diseaseprogressive liver diseaseportal systemic shunt superimposed acute liver disease progressive liver disease portal systemic shunt	hepatic encephalopathy and myxedema coma share clinical features : coma, ascites, anemia, impaired liver functions, and a metabolic electroencephalogram (eeg). hyperammonemia, a hallmark of hepatic encephalopathy, has also been described in hypothyroidism. differentiation between the 2 conditions, recognition of their possible coexistence, and the consequent therapeutic implications are of utmost importance. we describe a case of an 82-year - old woman with a history of mild chronic liver disease who presented with hyperammonemic coma unresponsive to conventional therapy. further investigation disclosed severe hypothyroidism. thyroid hormone replacement resulted in gain of consciousness and normalization of hyperammonemia. in patients with an elevated ammonia level, altered mental status, and liver disease, who do not have a clear inciting event for liver disease decompensation, overwhelming evidence of hepatic decompensation, or who do not respond to appropriate therapy for hepatic encephalopathy, hypothyroidism should be considered and evaluated.
acinar cell carcinoma, also known as acinic cell carcinoma, is a malignant epithelial neoplasm composed of cells with morphological resemblance to acinar cells and with evidence of exocrine enzyme production by the neoplastic cells. these carcinomas of the pancreas are rarely diagnosed pre - operatively, and surgical resection is the best treatment modality. median overall survival after successful resection is 36 months in reported studies. a brief review of clinical presentation, diagnosis and management is discussed. a 35-year old male patient presented in out patient department, with complaints of diarrhea for last 2 years and pain upper abdomen for last 15 days. there were no other associated symptoms, co - morbidities and he was nonalcoholic and non - smoker. general physical examination revealed no abnormality, and on abdomen examination there was a firm, nontender, 88 cm retroperitoneal lump, with well - defined margins, in epigastrc region and also occupying part of left hypochondrial and umbilical region, with no organomegaly and no free fluid. ultrasonography of the abdomen showed a large 7.76.48.4 cm size heterogeneously hypoechoic mass, involving the body of pancreas, while the head of the pancreas, liver, spleen and other organs were normal. contrast enhanced computed tomography showed a large, heterogeneously enhancing, well defined intrapancreatic lesion, of size 127.5 cm in body and tail region, with few small non - enhancing areas, indentation on posterior wall stomach, and loss of intervening fat plane (figure 1). there were no evidence of ascites, liver and peritoneal metastasis, and peripancreatic and retroperitoneal lymphadenopathy. oesopha - gogastroduodenoscopy showed the presence of extrinsic impression on the posterior wall of gastric body. colonoscopy and tumor marker ca 19 - 9 were normal. in view of a large mass involving the body of the pancreas and no evidence of metastasis, after adequate preoperative preparation intra - operatively, there was a large mass of size 128 cm arising from the body and part of tail of pancreas, abutting splenic vessels and transverse mesocolon while pancreatic head was normal. the mass was free from stomach, transverse colon, duodenum and spleen (figure 2), and there were no palpable lymph nodes, liver and peritoneal metastasis and ascites. on histopathological examination, grossly, well circumscribed grey brown soft tissue mass measuring 1288 cm, with multinodular external surface, and cut section was variegated with layers of hemorrhage and necrosis, and on microscopy, there was partly encapsulated tumor composed of cells arranged mainly in diffuse sheets, trabecular and focally in acinar pattern, at places tumor sheets are separated by thick fibrous septa and cells shows mild pleomorphism, round in shape, with indistinct cell membranes, moderate amount of granular eosinophilic cytoplasm, round to oval hyperchromatic nuclei (figure 3a). periodic acid - schiff (pas) staining with diastase showed granular cytoplasmic positivity (figure 3b), and immunohistochemistry demonstrated acinar enzymes, trypsin and chymotrypsin. post - operative period was uneventful and the patient received 6 cycles of chemotherapy (folfox regimen) and got discharged on post - operative day 18. acinar cell carcinoma is a rare pancreatic malignancy, and constitutes 1 - 2% of all exocrine pancreatic neoplasms in adults and 15% of those in pediatric age group of all pancreatic tumors. though acinar cells makeup the bulk of the pancreas, pancreatic neoplasms exhibiting predominantly acinar differentiation are rare. not more than 25% of the neoplastic cells exhibit endocrine or ductal components and cases with significant endocrine or ductal components (more than 25%) are regarded as mixed carcinomas. there is a slight male preponderance and mostly presents in 5 to 7 decades of life. acinar cell carcinomas arise more commonly from head though they may arise from any part of the pancreas. most commonly, acinar cell carcinomas present with non - specific symptoms like weight loss (52%), abdominal pain (32%), nausea and vomiting (20%), in contrast to ductal adenocarcinoma, acinar cell carcinoma rarely obstructs the common bile duct because they generally compress and do not infiltrate into adjacent structures, so jaundice is infrequent (12%). our patient presented with chronic diarrhea and upper abdominal pain. paraneoplastic syndrome may be the only presenting symptom in 15% of patients, and lipase hypersecretion syndrome characterized by subcutaneous fat necrosis, polyarthralgia and peripheral eosinophilia is a type of paraneoplastic syndrome associated with it. this syndrome is more commonly encountered in patients with hepatic metastasis, although occasionally this could be due to an extremely large organ - limited primary carcinoma. successful surgical removal of the neoplasm may result in the normalization of the serum lipase levels and resolution of the symptoms. serum glycoprotein markers (ca 19 - 9) are usually not elevated and a modest elevation in serum lipase levels may be detected in those patients without the lipase hypersecretion syndrome. analyzed cell lineage markers, p53 expression, and k - ras mutations of acinar cell carcinoma, demonstrating that they constitute an entity different from ductal adenocarcinoma or endocrine tumors. in recent molecular analysis of acinar cell carcinomas, it was found that there are abnormalities in the apc/catenin pathway similar to those found in colorectal cancer. for this reason, chemotherapeutic agents effective in pancreatic adenocarcinoma and colorectal carcinoma like gemcitabine, cisplatin, 5fu, leucovorin, oxaloplatin, irinotecan, capecitabine etc. are effective in acinar cell carcinoma, and we also gave our patient folfox regimen. four patterns of growth have been described : acinar, cellular, trabecular, and glandular. in our case, it was mixed acinar and trabecular pattern. acinar enzymes, especially trypsin, chymotrypsin are demonstrable by immunohistochemistry in essentially all cases. in most of the studies, approximately 50% cases presented with metastasis at the time of diagnosis and it is different from adenocarcinoma in that it has more indolent course with 5-year survival over 40%, with median survival 36 month, and the genetic alteration of ductal adenocarcinoma like kras, tp53, p16, or smad4 are absent in acinar cell carcinomas. in conclusion, acinar cell carcinomas are rare pancreatic tumors, present mostly with non - specific symptoms, difficult to diagnose pre - operatively, surgery is the mainstay of therapy, and there are no standard chemotherapy regimens and prognosis is better than adenocarcinomas.	acinar cell carcinoma of the pancreas is a rare pancreatic malignancy, constituting only 1 - 2% of all the pancreatic tumors. a young adult male presented with chronic diarrhea and upper abdominal pain, on investigations was found to have a large pancreatic tumor of size 127.5 cm involving the body of the pancreas. pancreatic body and tail resection with splenectomy was done and final histopathological examination showed acinar cell carcinoma of the pancreas. prognosis of acinar cell carcinoma is better than adenocarcinoma of the pancreas.
	we performed a cross - sectional study that included 100 hiv - infected japanese men without hemophilia to examine the influence of smoking on hiv infection. history of smoking was obtained using a questionnaire. the percentage of current smokers was 40 % and was the highest (50 %) among men in their forties. the mean brinkman index (bi, number of cigarettes smoked per day multiplied by years of smoking) was 450. the percentage of patients with a bi 600 was significantly higher in patients with an aids - defining event than in those without an aids - defining event. a bi 600 was associated with an aids - defining event. reducing smoking appears to be critical to enhancing disease management efforts in japanese men with hiv.
the basal forebrain cholinergic neurons (bfcn) provide the major cholinergic innervation to the hippocampus and neocortex, playing a role in cognition and attention behaviors through the release of the neurotransmitter acetylcholine. bfcn are located in the medial septum, diagonal band of broca, nucleus basalis of meynert and striatum. they are found to be massively degenerated in late stages of sporadic alzheimer 's disease (ad), one of the most diffuse and lethal disease of the elderly. however, in contrast with the so called cholinergic hypothesis, ad can not be considered as a generalized brain cholinergic disease (mesulam, 2004). indeed, the cholinergic system undergoes only a mild reduction of synaptic density and a partial atrophy at the ad onset, while frank bfcn degeneration and death require more than a decade to appear (mesulam, 2004). functional bfcn synapses relay on continuous and activity - dependent release of nerve growth factor (ngf) by cortical and hippocampal neurons (iulita and cuello, 2014). ngf binds to two classes of cell surface receptors localized at the bfcn terminals : the specific ngf receptor tyrosine kinase a (trka) and the common neurotrophic receptor p75. the ngf signal is retrogradely conveyed from axons and dendrites toward the nucleus of bfcn, where it modulates cholinergic gene expression. the requirement of an active ngf / trka pathway in forebrain - related cognition is confirmed by the positive correlation between trka levels and mini - mental status examination scores. as mentioned above, a number of experimental results prompt the perturbation of the ngf pathway as an early event in ad pathology. in fact, alterations of the ngf / trka signaling system correlate well, and even more robustly than the amyloid load, with cognitive deficits in mci and in its progression toward ad. moreover, single bfcn expression analysis indicates that trka mrna is reduced in mci, and suggests that decreased neurotrophin responsiveness may be an early ad biomarker (mufson., 2012). since the pro - apoptotic ngf precursor (prongf) increases while trka levels diminish in the ad forebrain, it is conceivable that degenerative pathways may override ngf - trka survival signals during pre - sympthomatic ad (mufson., 2012). further, experimental findings from animal and cellular models indicate that the impairment of the ngf signaling system may be a critical event in the manifestation of this pathology. accordingly, in vitro ngf deprivation in pc12-derived and primary hippocampal neurons alzheimer 's like molecular syndrome with both amyloid and tau accumulation (calissano., 2010). moreover, antibody - mediated neutralization of ngf promotes the appearance of histopathological signs typical of ad, including amyloid generation and neuronal deficits in the ad11 mouse model (ruberti., 2000). thus, it would seem reasonable to antagonize basal forebrain dysfunctions in ad by exogenous ngf administration. in line with this, intracerebral ngf supply has been found beneficial to cholinergic neurons and related behavior in rodents. in particular, nasal administration of ngf modulates secretases levels and reduces amyloid burden in app / ps1 transgenic mice (yang., 2014). of note, ngf gene therapy has been attempted in a phase 1 clinical trial, which showed high tolerability and lack of side effects in ad patients. also, the results of a 10 years long study confirmed safety and efficacy of the ngf therapy, and reported long lasting brain responsivity to ngf in terms of activation of functional markers, hypertrophy, and axonal sprouting (tuszinsky., 2015). as less invasive alternatives to intracerebral stereotaxic ngf delivery, the ngf administration via the ocular or nasal routes has also been performed in rodents and promoted cholinergic system neuroprotection. all together, these findings underline the importance of a proper homeostatic regulation of the neurotrophic pathway in the early phase ad and pinpoint the feasibility of ngf therapy, claiming the need for efficient, safe and long - lasting therapeutic approaches for the ngf treatment of ad. activity - dependent release of ngf from cortical and hippocampal neurons has been demonstrated to sustain the cholinergic tone on bfcn target neurons via the muscarinic receptors 1 (m1). ngf has been thought to maintain neuronal homeostasis and forebrain - related cognition mainly through this interplay. in fact, m1 activation induces the physiological cleavage of app to generate soluble app (sapp), which is neuroprotective per se, and it is also a potent inhibitor of the enzyme responsible for the amyloidogenic app cleavage, the beta secretase 1 (bace1). whether ngf directly regulates app metabolism in bfcn indeed, we recently demonstrated that the ngf signaling pathway is able to modulate app processing in bfcn both in vitro and in vivo (triaca., 2016). we showed that stimulation of primary cholinergic septal neurons with ngf promotes binding of the ngf receptor trka with app and the preferential app trafficking to the golgi compartment, where app binding to and cleavage by bace1 is hampered. as a result, the levels of bace - generated app fragments, like soluble app (sapp), c - terminal fragment (ctf) and beta amyloid (142), are strongly reduced. in particular, we observed that binding of trka to app is facilitated by ngf through the reduction of app phosphorylation at the threonine 668 (t668) residue of its cytosolic tail. in fact, co - localization and co - immunoprecipitation analyses showed that trka fails to bind app molecules phosphorylated at t668 (app), suggesting that app phosphorylation prevents app binding to trka as already observed for another app interactor, namely fe65. app phosphorylation at t668 is a post - translational modification known to facilitate app cleavage by bace1 and amyloid generation, and it was proposed as target for ad therapy (lee., 2003). thus, ngf exerts its anti - amyloidogenic action by lowering the fraction of app molecules, favoring trka - app interaction and the subsequent app processing along the anti - amyloidogenic route. the early downstream molecular players involved in the control of app metabolism have also been investigated. upon ngf binding, trka activation promotes the phosphorylation of the isoform c of the sh2 containing sequence (shc), the early trka adaptor expressed in adult bfcn. afterward, shcc activation inhibits the p54 isoform of the c - jun n - terminal kinase p54 (jnk), a well known ser / thr app kinase, thus reducing app levels, and promoting app - trka binding (figure 1). a schematic model illustrating amyloid precursor protein (app) metabolism control by the nerve growth factor / tyrosine kinasea ngf / trka signaling system in healthy basal forebrain cholinergic neurons (bfcn) (anti - amyloidogenic route, left), and the consequences of its perturbation in alzheimer 's disease (amyloidogenic route, right). anti - amyloidogenic route., trka trans - phosphorylation takes place, inducing trka phosphorylation at the tyrosine 490 (y490) residue and trka docking of the signaling adaptor sh2 containing sequence c (shcc). once activated, shcc inhibits c - jun n - terminal kinase (jnk), a ser / thr app kinase thus hindering the phosphorylation of app at the threonine residue 668 (t668). since trka is able to bind only app molecules not phosphorylated at t668, the reduction of app levels induced by ngf promotes 1) app - trka binding, 2) subsequent trka - mediated trafficking of app to the golgi compartment and to the plasmamembrane, and 3) preferential cleavage of app by the neuronal alpha secretases 1017 (adam1017) through the physiological pathway. reduced availability of mature ngf, and/or expression levels of trka affect app metabolism in bfcn. in fact, disturbances in the anti - amyloidogenic ngf / trka - shcc signaling pathway grant the activation of jnk by pro - apoptotic signals, resulting in augmented app levels and disruption of app interaction with trka in favor of beta secretase 1 (bace1) binding and cleavage of app along the amyloidogenic pathway., 2016. the app - trka interaction seems to have a pathological significance in ad. in fact, app - trka interaction is specifically lost in ad affected tissues, like the hippocampus, while it seems to be preserved in the ad cerebellum, as well as in the hippocampus of patients affected by other neurodegenerative diseases, like huntington 's disease. a deeper analysis of the app - trka interaction in bfcn in vitro and in vivo by proximity ligation assay (pla) and bimolecular fluorescence complementation (bifc) is currently ongoing in our group. altogether, these findings suggest that the ngf system maintains amyloid levels within the physiological range in healthy bfcn by modulating app processing by bace1. based on reduced trka and/or ngf levels observed in mci and early ad, it is tempting to speculate that disturbances in attention and cognition may result from the perturbation of the ngf - trka - shcc pathway in bfcn, inducing and/or contributing to synaptic deficits of their hippocampal and cortical target neurons. it is well known that bfcn are more vulnerable to ad, as compared to those located in the cerebellum. higher forebrain susceptibility to intraneuronal amyloid accumulation has been suggested to account for this difference. intraneuronal amyloid accumulation has been extensively demonstrated to occur during brain ageing and in ad pathology in the bfcn of mice, monkeys and humans by the geula lab (baker - nigh., 2015). a substantial increase of intraneuronal amyloid long before plaques formation has also been reported in 3xfad transgenic mice (la ferla., 2007). while intraneuronal amyloid is neuroprotective against oxidative stress at physiological levels (picomolar), higher concentrations therefore, intraneuronal amyloid accumulation has been prospected as a good predictor of synaptic and neuronal loss (bayer and wirths, 2010). as elegantly demonstrated by laferla. (2007), the newly generated amyloid first appears inside neurons and afterwards outside the cells, suggesting that intraneuronally generated amyloid can be released into the extracellular space causing plaque deposition in 3xfad transgenic mice. novel findings from clinical examinations, amyloid imaging, and functional mri provide evidence that not only neocortical regions, but also subcortical areas of the basal forebrain (e.g., striatum) show amyloid accumulation and neurodegeneration at the pre - symptomatic ad stage. accordingly, early ad pathology is characterized by signs and symptoms of dysfunctional subcortical circuits (shinohara., 2014). in line with this, the study of amyloid accumulation in the bfcn of mouse models lacking the mature ngf signaling will be instrumental in the pathological and molecular profiling of the ad onset. based on the relevance of ngf signaling in the physiological control of app processing in the basal forebrain, it can be hypothesized that lack of neurotrophic support may boost amyloid generation and intracellular accumulation in bfcn, thus promoting the initial synaptic disturbances seen in mci and early ad. here, we prospect that upon ngf withdrawal cholinergic neurons may primarily contribute to ad pathology and affect target neurons in the cortex and hippocampus by generating and releasing amyloid, possibly through the exosomal and/or synaptic routes. on the other hand, the newly generated amyloid is able to inhibit the endocytosis of the ngf / trka complex at the cholinergic terminals, in a negative feedback loop which settles the ad onset (kim., 2016 ; xu., 2016 once age - related events (oxidative stress, astrogliosis, reduced amyloid clearance) occur, they compromise the brain buffering capacity and determine the overt neuronal loss of bfcn and their targets typical of late stage ad. the fine analysis of the spatio - temporal sequence of amyloid appearance in the ad brain will hopefully provide important insights into the pathological drivers of this devastating neurodegenerative disease of the elderly, paving the way for novel targeted approaches in ad therapy.	the current idea behind brain pathology is that disease is initiated by mild disturbances of common physiological processes. overtime, the disruption of the neuronal homeostasis will determine irreversible degeneration and neuronal apoptosis. this could be also true in the case of nerve growth factor (ngf) alterations in sporadic alzheimer 's disease (ad), an age - related pathology characterized by cholinergic loss, amyloid plaques and neurofibrillary tangles. in fact, the pathway activated by ngf, a key neurotrophin for the metabolism of basal forebrain cholinergic neurons (bfcn), is one of the first homeostatic systems affected in prodromal ad. ngf signaling dysfunctions have been thought for decades to occur in ad late stages, as a mere consequence of amyloid - driven disruption of the retrograde axonal transport of neurotrophins to bfcn. nowadays, a wealth of knowledge is potentially opening a new scenario : ngf signaling impairment occurs at the onset of ad and correlates better than amyloid load with cognitive decline. the recent acceleration in the characterization of anatomical, functional and molecular profiles of early ad is aimed at maximizing the efficacy of existing treatments and setting novel therapies. accordingly, the elucidation of the molecular events underlying app metabolism regulation by the ngf pathway in the septo - hippocampal system is crucial for the identification of new target molecules to slow and eventually halt mild cognitive impairment (mci) and its progression toward ad.
renal autotransplantation is a method of removing kidney from its place of origin, repairing it and transplanting it in another location of the body (most commonly, the iliac fossa) of the same patient. this procedure was first performed by hardy in 1963. since hardy 's landmark surgery for management of a high ureteral injury, renal autotransplantation has been described in the treatment of renal arterial disease, renal cell carcinoma, nephrolithiasis etc. it has been demonstrated that tumour size (> 4 cm) identified on computerised tomography (ct) scan, is the most reliable predictor of rupture. here we describe the anaesthetic challenges in a bilateral ex - vivo nephron sparing surgery with autotransplantation for giant angiomyolipoma. a 28-year - old unmarried female weighing 55 kg presented with lower abdominal discomfort and vague lump in the abdomen of 5 years duration. blood investigations were normal and contrast enhanced abdominal ct with angiogram showed bilateral highly vascular huge renal masses suggestive of angiomyolipoma [figure 1 ]. she was taken up for right radical nephrectomy, bench surgery and autotransplantation after adequate pre - operative preparation. pre - operative computed tomography angiogram - highly vascular angiomyolipoma we instituted general anaesthesia supplemented with low thoracic epidural at t11 - 12 interspace and epidural analgesia was maintained with 0.2% ropivacaine and fentanyl 2 mcg / ml at the rate of 4 - 6 ml / h. anaesthesia was induced with glycopyrrolate 0.2 mg, midazolam 1 mg, ramosetron 0.3 mg, fentanyl 100 mcg and propofol 100 mg. intubation was facilitated with 100 mg succinylcholine and anaesthesia was maintained with n2o, o2, isoflurane and atracurium. electrocardiogram, pulse oximetry, invasive blood pressure, end - tidal carbon dioxide, central venous pressure (cvp), core temperature and hourly urine output were monitored. abdomen was entered through a right thoracoabdominal incision [figure 2 ], excising the tenth rib and splitting the diaphragm. right kidney with the mass (30 cm 15 cm) was removed and cannulated for cooling with the histidine tryptophan ketoglutarate solution. the warm ischemia time was 1 min and 45 s. bench dissection was carried out and kidney was autotransplanted. cold ischemia time was 4 h. during this period, the anaesthetic goal was to maintain mild hypertension. patient had massive blood loss approximately 5 l, which was corrected with five units of packed cells, four units of fresh frozen plasma, 9 l of crystalloids and 1 l of colloid. she had continuous oozing from the wound site suggestive of coagulopathy due to massive blood loss and transfusion which was corrected with further transfusion of blood components (two units of packed cells, three units of fresh frozen plasma, four units of platelets and four units of cryoprecipitate). she was haemodynamically stable and coagulopathy was corrected by the second post - operative day and she was weaned off inotropic and ventilatory support. after 4 weeks, she was posted for the left nephrectomy with bench surgery and autotransplantation as she had a large tumour (26 cm 18 cm) in the left kidney with high risk of bleeding. renal doppler showed suboptimal function of right transplanted kidney. only a single arcuate artery in the lower pole cortex picked up doppler flow with increased resistance index. the surgical technique and induction of anaesthesia were similar to what was used for the right nephrectomy. patient was adequately hydrated before nephrectomy with 3 l of crystalloids to maintain adequate urine output. warm ischemia time was 2 min 13 s. cold ischaemia time was 2 h 48 min. after the nephrectomy, fluids were restricted with a goal to maintain cvp of 6 - 7 cm of water. before the release of the cross clamp after transplantation, cvp was increased to 16 - 18 cm of h2o. the target blood pressures were systolic more than 140 mm hg, mean arterial pressures more than 95 mm hg and the diastolic pressure more than 85 mm hg as was practiced in renal allotransplantation. frusemide 200 mg and sodium bicarbonate 25 ml were administered and urine output was adequate after reperfusion. blood loss was around 1.5 - 2 l. she was infused nine litres of crystalloids, 500 ml colloid and two units of packed cells. the urine output was 3.6 l. at the time of discharge her creatinine was 1.4 mg / dl and it was 1.5 mg / dl on day 30. the follow - up angiogram after 1 month showed a well - perfused kidney on the left side. ex - vivo excision of the tumour and autotransplantation is a feasible option for bilateral giant renal lesions in select cases, thus avoiding the morbidity associated with bilateral nephrectomy and renal replacement therapy. it is a technically demanding procedure and is contraindicated in severe occlusive atherosclerosis of the iliac arteries. renal autotransplant is a two - step procedure ; first the kidney is removed, dissected extracorporeally and then transplanted. surgical approach in removing the kidney is similar to that of living donor nephrectomy. the problems that we had during the right nephrectomy were massive blood loss due to the highly vascular large tumour and maintenance of adequate renal perfusion. once the autotransplantation was completed it was important to maintain adequate intravascular volume with colloids or blood to ensure well - perfused kidney. cvp was maintained at 16 - 18 cm of water and the mean arterial, systolic and diastolic pressures were maintained above 95 mm hg, 140 mm hg and 85 mm hg respectively, with risk of increased blood loss. we had to start the patient on dopamine and dobutamine to maintain the blood pressures. intravenous mannitol minimises ischemic injury to the kidney and hasten the restoration of renal function. for the second autotransplant the problems we anticipated were a large angiomyolipoma which could bleed profusely making maintenance of cvp and blood pressures difficult. also, the operated kidney had suboptimal function complicating fluid management. after discussion with the surgeon, we decided to manage the patient as a donor before the nephrectomy and administered fluids as for a donor for allotransplant. after the nephrectomy, as the patient was anepheric we decided to treat the patient as an allotransplant recipient. a targeted approach, with restriction of fluids and replacement of blood loss was practiced to maintain the cvp at 6 - 7 cm h2o during bench dissection. during the final stages of bench dissection, cvp was raised to 16 - 18 cm of h2o, thus enabling the patient to be adequately filled up during reperfusion with achievement of the target mean arterial, systolic and diastolic pressures. flower vase thoracoabdominal incision was used [figure 2 ] and analgesia was maintained with a low thoracic epidural, intravenous fentanyl, tramadol and paracetamol. renal bilateral ex - vivo nephron sparing surgery with autotransplantation is a highly invasive surgery, which demands proper intraoperative fluid management and maintenance of adequate urine output. invasive monitoring is preferred to achieve this goal and enable smooth patient recovery and preservation of renal function.	extracorporeal work bench surgery with subsequent autotransplantation is a challenge from both anaesthetic and surgical point of view when performed bilaterally or in a solitary kidney. a 28-year - old female with bilateral giant angiomyolipoma of kidneys was taken up for renal autotransplantation. patient had a huge tumour, which was the largest reported exophytic tumour to be excised by this technique. both kidneys were operated at an interval of 1 month, under combined general and epidural anaesthesia. anaesthetic challenges faced during the procedure were maintenance of adequate perfusion of the grafted kidneys, containment of massive blood loss and coagulopathy during the perioperative period. patient recovered in due course with functioning autotransplanted kidney. a careful pre - operative preparation with intraoperative maintenance of adequate blood volume and blood pressure is the key for graft survival.
although current cross - sectional data suggest that most americans, including children and adolescents, consume a significant portion of their daily energy as snacks, snacking remains a poorly understood behavior. there is little information on how and why individuals and families select snacks, and no consistent definition of snacks or snacking used by most consumers or even the research community. many studies, including the national health and nutrition examination surveys, rely on participants to define snacks themselves. while some individuals define snacks as an eating occasion between meals, others define snacks based on the type of food consumed, location of food consumption, or time of day of consumption. unlike other eating occasion labels like breakfast, lunch, or dinner, snacks commonly describes a type of food as well as an eating occasion. the 2015 dietary guidelines for americans, for instance, caution against excessive consumption of snacks, with regard to the type of food, because they add sugars and saturated fat to the american diet, but they recommend snacks as an eating occasion, suggesting carrots with hummus as a sample snack meal. based on consumer definitions, however, americans receive a quarter of their daily energy from snacks. the 2015 scientific report of the dietary guidelines committee states that 96% of the us population over the age of 2 years eats at least one snack every day and that daily consumption of 2 to 3 snacks is even more common. the results of 2 recent studies suggest that the type of snack, rather than the frequency of consuming snacks, is the most important determinant of whether snack consumption is associated with adiposity, diet quality, or body mass index. however, the term snacking is still often associated with the consumption of foods high in saturated fat, sugar, and sodium, commonly referred to as snack foods. while snack foods are often associated with nutrients to limit, like most foods, snacks also include nutrients to encourage. yet guidance about overall nutrient composition and the nutrient density of snacks remains largely unavailable. food labels, for example, draw consumer attention to the calorie and fat content (perceived by many to be less healthful nutrients) at the top of the label but not to the same food s other nutrients such as calcium, potassium, and fiber, listed further down the label. consumers who read labels, including adults who purchase snacks for their children, tend to read only the first 5 components (servings, calories, total fat, saturated fat, and trans fat) of the nutrition facts label, none of which are nutrients to encourage. this may explain why label reading does not necessarily lead to the selection of foods high in nutrients to encourage. comprehensive dietary guidance about common snack choices based on nutrient density would be useful for different stakeholders. with this guidance, parents could more easily identify healthful snacks for their children, clinicians would have reliable information for counseling patients about snacking and dietary needs, and researchers would be able to assess more easily the impact of dietary trends or interventions that involve snacking. the purpose of our study was to quantify the nutrient density of commonly consumed snacks using the nutrient - rich foods (nrf) index, and therefore fill an important need by showcasing a way to assess the nutritional value of snacks, which make up a large part of the diets of children and families. for the purposes of this article, snacks are defined as food or caloric beverages consumed between regular meals (breakfast, lunch, and dinner). the nrf index assigns scores to foods based on their nutrients to encourage (protein, calcium, vitamin d, potassium, magnesium, iron, vitamin a, vitamin c, vitamin e, and fiber) and nutrients to limit (sodium, saturated fat, and total sugar). we obtained data on the most commonly consumed snack categories in the united states from the 2014 national eating trends (net) survey administered in paper form by the npd group, a market research company. the net survey includes data from roughly 5000 individuals annually, 23% of whom are children. net participants are recruited from a national mail panel, and the main food preparer / purchaser in each household (panelist) records the food and beverage consumption of all household members for a 2-week period. panelists could record up to 3 snacks (defined as a between meal eating occasion) and up to 3 meals per individual per day. although families enrolled in the net are nationally representative in many ways, including geographic distribution, survey participants are, in general, better educated than americans as a whole. for example, roughly 46% of main food preparers / purchasers have a college degree compared with about 33% of americans as whole. hispanics and african americans are also underrepresented in the sample compared to the us population, making up just 7.9% and 5.8% of participants, respectively, compared with 13.3% and 17.6% of americans as a whole. next, we obtained brand information for the 3 market leaders in each snack category identified from the net based on 2014 - 2015 sales data from information resources, inc (iri ; http://www.iriworldwide.com/en-us). table 1 does not list nonbranded products (fruit and some varieties of milk). the nonbranded types of milk most commonly consumed for snacks were 2% milk and whole milk, and the most popular types of fruit selected for snacks were apples, bananas, and grapes. table 1 also does not include private label top sellers. if 1 of the 3 market leaders was identified as private label in the iri data, a generic version of the product (ie, chocolate chip cookies) was selected from the nutrient database (described below) in lieu of a branded product. nutrient data for snacks were obtained from the nutrition data system for research software, version 2014, developed by the nutrition coordinating center at the university of minnesota, minneapolis, mn. when nutrient details for specific branded foods were not available in this database, we obtained nutrient information by contacting manufacturers directly. we calculated nrf scores for each product and for each snack food category using microsoft excel (version 2010, microsoft, inc, redmond, wa). a few food items included in this analysis, namely, diet cola, sugar free gum, and brewed tea (from tea bags), contain no calories or very few calories in each serving and were excluded from our calculations. finally, we calculated nutrient - density scores for each food. this study uses a modified version of the nrf index 9.3, to which we have added vitamin d (listed as a nutrient to encourage in the 2010 and 2015 dietary guidelines for americans). we have designated this vitamin d augmented version of the nrf index as nrf 10.3. first, for each 100 kcal of a specific food, the amount of each nutrient to encourage was expressed as a percentage of its daily recommended value. next, for 100 kcal of the same food, the amount of each nutrient to limit was calculated as a percentage of the recommended limit. the nrf index was then calculated as the sum of the values for nutrients to encourage minus the sum of the values of nutrients to limit. it is often difficult or impossible based on common data sources to accurately distinguish between added and total sugars for many snack foods. the daily reference value used here (125 g) was adopted from an overview of the nutrient - rich foods index. fruit, selected as a snack by 48% of net respondents in the 2-week survey period, was the most popular snack and had an nrf index score of 30.1. cookies, chips, and ice cream followed in popularity, selected by 44%, 33%, and 33%, respectively (table 3). among the most popular snack categories, nrf scores varied from 17 to 55 (table 4). yogurt, milk, and fruit were the most nutrient - dense snack categories, while ice cream, pies and cakes, and carbonated regular soft drinks were the most nutrient - poor snacks. the median nrf score for all snack options assessed was 6.0. with the breadth of scores (17 to 76) for individual snacks, while chips are commonly considered a food high in nutrients to limit, potatoes naturally contain potassium, magnesium, fiber, and vitamin c, and the oil used in chip production adds vitamin e. in addition, chip companies have transitioned to vegetable oils in recent years, limiting saturated fat content. the snacks in the categories with the highest nutrient density, namely, yogurt and milk, contain high amounts of nutrients to encourage, especially protein, calcium, potassium, vitamin d, and magnesium, with relatively small amounts of nutrients to limit (saturated fat, total sugars, and sodium) in a 100 kcal serving. yogurt scored higher than milk in this analysis because the leading yogurt products are all nonfat, which has less saturated fat than the market - leading milk varieties (2% and whole). both yogurt and milk do have relatively low amounts of iron, vitamin a, vitamin c, vitamin e, and fiber. fruit, the third most nutrient - dense category, contains high amounts of vitamin c, fiber, potassium, and magnesium, and relatively low amounts of protein, calcium, vitamin d, vitamin a, vitamin e, and iron. compared with yogurt and milk, fruit has a higher total sugar content (a nutrient to limit in this analysis), which decreased its nrf score. the most nutrient - dense snacks, milk and yogurt, were also the least frequently consumed. only 21% of consumers recorded milk for a snack, and a mere 14% of respondents ate yogurt. several of the foods evaluated in this analysis, including all of the yogurt products, milks, fruits, nuts and seeds, and potato chips had relatively high nrf index scores, indicating nutrient density. other frequently selected snacks including soft drinks, pies and cakes, ice cream, and cookies had negative nrf scores and, therefore, low nutrient density. flavored milk, for example, contains more added and total sugars than plain milk, but is also rich in calcium and vitamin d, both of which are nutrients to encourage. the 2015 dietary guidelines for americans recommend choosing nutrient - dense foods and beverages and then define these foods and beverages as containing little or no solid fats and added sugars, refined starches, and sodium but mention no specific nutrients to encourage. evaluating the nutritional value of any food based only on its contribution of nutrients to limit is unreasonable. our analysis provides a more balanced analysis of the nutritional value of commonly consumed snacks but is prone to several limitations. weighing nutrients equally as in the nrf index calculations may not be a valid method for assessing overall nutritional value. it is not clear to what degree each nutrient to encourage or nutrient to limit contributes to or detracts from health or the overall nutritional value of a food. the nrf index is a useful, though imperfect, tool for a more balanced understanding of commonly consumed snacks in the united states. physicians, dietitians, and other clinicians faced with the challenging task of providing brief counseling on diet and exercise to children and their parents could use the nrf index to discuss specific snack foods based on their overall nutrient profiles. jh : ms. hess conducted the data analysis for this publication with the assistance of the coauthors. she wrote the 1st draft of the paper and made revisions as suggested by the coauthors. rao made significant revisions to the paper and supplied information on the use of nrf in clinical settings.	background : although americans receive almost a quarter of their daily energy from snacks, snacking remains a poorly defined and understood eating occasion. however, there is little dietary guidance about choosing snacks. families, clinicians, and researchers need a comprehensive approach to assessing their nutritional value. objective : to quantify and compare the nutrient density of commonly consumed snacks by their overall nutrient profiles using the nutrient - rich foods (nrf) index 10.3. methods : nrf index scores were calculated for the top 3 selling products (based on 2014 market research data) in different snack categories. these nrf scores were averaged to provide an overall nutrient - density score for each category. results : based on nrf scores, yogurt (55.3), milk (52.5), and fruit (30.1) emerged as the most nutrient - dense snacks. ice cream (4.4), pies and cakes (11.1), and carbonated soft drinks (17.2) emerged as the most nutrient - poor snacks. conclusions : the nrf index is a useful tool for assessing the overall nutritional value of snacks based on nutrients to limit and nutrients to encourage.
colonic diverticulosis is a very frequent disease in the west. in 40- to 50-year - old persons, its incidence increases with age, reaching over 50% in persons > 70. in most cases (80%), the disease is asymptomatic (diverticulosis) and has a mild functional clinical scenario of dyscinesia. a few patients, about 10% to 20%, have simplex acute or complicated diverticulitis (perforation, abscess, fistula, hemorrhage, circumscribed or generalized peritonitis). laparoscopy has been used to treat diverticular disease, and currently it is a widely accepted and used procedure. the laparoscopic approach is not commonly used to treat neoplastic pathology of the colon - rectus ; however, its use is not restricted in treating phlogistic diseases. in fact, data about the feasibility and advantages of the laparoscopic approach for treating diverticular disease are reported in the literature. greater postoperative comfort, with a reduction in pain and lower need for analgesics, a more rapid resumption of intestinal function, a shorter hospital stay, and a more rapid return to social activity between january 1999 and january 2001, 5 patients underwent elective treatment with the laparoscopic approach for uncomplicated diverticulitis. the phases of the procedure were the usual : mobilization of the left colon, isolation and section of the inferior mesenteric artery, sovrapubic incision for operative specimen removal, preparation of the proximal stump, closure of the distal stump with endo - gia, and end - to - end intracorporeal coloproctostomy with a circular stapler. in the preceding period (september 1997 through december 1998), the surgical indication, always elective, was determined on the second admission for acute and obviously uncomplicated diverticulitis. the classifications of diverticular disease according to hinchey and wexner are shown in table 1. classification of diverticular disease according to hinchey and wexner the symptomatology is characterized by fever, leukocytosis, nausea, vomiting, pain, and medium - degree contracture of defense in the inferior left abdominal quadrant. a mass in the lower abdominal quadrants or an extensive contracture of defense, except for the left inferior quadrant if either is present, emergency surgery should be performed because of the presence of an abscess or circumscribed or generalized peritonitis from a micro or macro diverticular perforation. the elements of the clinical scenario of our patients enrolled in the study are outlined in table 2. we compared the results of the surgical treatment in both groups of patients (table 3). the number of patients and the demographic data (sex, age) are similar. postoperative morbidity (anastomotic dehiscences, wound infections, bronchopneumonic infiltrates) and mortality (within 30 days) are the same for both groups. on the contrary, the following elements are in evidence : a more rapid resumption of bowel movement and alimentation, with a precocious ambulation, and a shorter hospital stay in the patients who underwent the laparoscopic intervention. as to these facts,, we can affirm that, even without statistical validation, on the basis of the results obtained, with the same incidence of morbidity, the elective laparoscopic approach to colectomy for diverticulitis has a more rapid and comfortable postoperative course. surgery has a pre - eminent role on the general therapeutic plan when a benign, uncomplicated pathology is to be treated. we think that surgery should not be performed to treat simple diverticulitis when no inflammatory episodes have occurred. the appearance of acute diverticulitis in the fourth, fifth, and sixth decades represents a further favorable element for surgical indication. moreover, episodes of acute diverticulitis are considered uncomplicated if medical therapy, begun early, leads to improvement in the clinical scenario within 48 hours. in our opinion, medical therapy must be effective quickly, resulting in improved symptoms within 48 hours. with the persistence or worsening of symptoms the medical therapy used is based on cessation of solid and liquid food intake, on absolute intestinal rest, on hydro - electrolyte reintegration, and on the use of broadspectrum parenteral antibiotics and analgesics. the clinical scenario must be evaluated quantitatively and qualitatively because the same signs with different degrees of expression can change the indication from medical therapy to urgent surgical therapy. moreover, comparison of the 2 surgical approaches (laparoscopic - assisted vs. open) must not improperly influence the choice between surgical and medical therapy, in the symptomatic but uncomplicated phase of the disease. surgery, almost always performed urgently, is necessary in cases of complicated acute diverticulitis, such as pericolic abscess, perforation, circumscribed or generalized peritonitis, and fistulization. however, even in these cases, the possibility of a laparoscopic approach can be considered. our cases fall within stage i of the hinchey - wexner classification, both in the laparoscopic and open exploration groups, excluding the cases of complicated diverticulitis (abscess, fistula, perforation, generalized peritonitis) in this study. in our opinion, great effort must be made not to change the clinical criteria for the indication because of the sometimes good results of the laparoscopic approach. then, the question that must be answered is : when must we operate on a patient when diverticulosis becomes symptomatic without significant complications. the following characteristics should be present : 2 consecutive attacks of acute diverticulitis (clinically controlled) ; adequate time interval between the 2 attacks (1 to 2 months) with appropriate medical - dietetic therapy, but not followed by stable improvement ; age < 60 years. the immediate postoperative results are to be examined in terms of operative morbidity and mortality and other possible advantages of one approach over another. the results of the stable correction of the pathology are not in discussion, because the intervention is identical in both procedures, so this result is related to the correctness of the indication. the immediate results can be evaluated based on the following criteria, and compared with those of the traditional open technique : operative time and index of conversion, resumption of bowel movements and alimentation, ambulation, hospital stay, use of analgesics in the postoperative phase (postoperative comfort), postoperative morbidity (anastomotic dehiscences, bronchopneumonic infiltrates, infections of the operative wound or of the trocar incisions, thrombo - embolic accidents), operative mortality. we think that, even with a longer operative time in comparison with the conventional approach and the necessity for adequate training in the laparoscopic technique, the best quality of the postoperative course and similar morbidity are evidence favorable to the laparoscopic procedure. at present, the routine indication of laparoscopy for benign colic pathologies is considered correct and effective. in fact, the advantages of the mini - invasive approach are incontestable, because of less postoperative pain, less parietal trauma, and the disappearance, after a while, of complications like postoperative laparocele. the resumption of intestinal function with a more precocious return to alimentation surely represents a great advantage, together with the rapid ambulation, a shorter and more comfortable postoperative stay, and a more rapid resumption of social activities. in this analysis, operative morbidity needs careful evaluation, and it assumes great importance in the treatment of a benign, uncomplicated pathology. data reported in the literature confirm that the occurrence of anastomotic dehiscences is similar in both the laparoscopic and open procedures. the remaining elements of postoperative morbidity are the same in the 2 procedures ; moreover, we must consider that the precocious ambulation after laparoscopic procedures allows a sort of protection from both the bronchopneumonic affects and thromboembolic accidents. the limits of the indication for laparoscopic surgery in the treatment of diverticular disease are the object of investigation at present. in acute, uncomplicated diverticulitis, the importance of a correct surgical indication and an adequate selection of the patients to undergo laparoscopic intervention is evident, so the surgical procedure is standardized. in an emergency, it is still difficult to define unequivocally the criteria for the choice of either the laparoscopic or the open approach to treat the complications of diverticular disease. the indication for the laparoscopic intervention for complicated diverticulitis is still an argument in evolution. in complicated diverticulitis (abscess, fistula, generalized peritonitis), the incidence of conversion is still very high : according to the literature, it should reach 61% versus 14% in acute uncomplicated diverticulitis. probably in the future, the laparoscopic indications will be very widespread even for complicated diverticulitis. the advantages of the laparoscopic approach in benign colonic pathology are represented by a shorter and more comfortable postoperative period with the minor use of analgesics, more rapid ambulation, and early resumption of alimentation and bowel activity. this study contributes to the literature by demonstrating the feasibility and safety of the laparoscopic - assisted left colectomy in uncomplicated diverticulitis.	objectives : the aim of this study was to evaluate the safety and effectiveness of laparoscopic - assisted sigmoid colectomy for diverticulitis and to assess its postoperative advantages.methods:from 1999 to 2001, 5 patients were selectively operated on with a laparoscopic - assisted procedure for uncomplicated sigmoid diverticulitis. in the preceding period (september 1997 through december 1998), 4 patients underwent open procedures for the same pathology. the surgical indication with the same criteria was restrictive : at least 2 acute episodes had occurred that were treated with hospital admission and that were separated by an adequate period (2 months) of medical therapy.results:no conversions of laparoscopy to an open procedure were necessary. age, sex, weight, morbidity, and mortality were similar between the 2 groups. operative time was 180 minutes for laparoscopy and 120 minutes for laparotomy. postoperative resumption of peristalsis was 24 hours versus 4 days, resumption of alimentation was on the second postoperative day versus the fifth postoperative day, and hospital stay was 7 days versus 12 days for laparoscopy and laparotomy, respectively.conclusion:this study shows the feasibility and the advantages of elective laparoscopic - assisted colonic re - section for uncomplicated sigmoid diverticulitis. the advantages of the laparoscopic approach are the lower need for analgesics and the more precocious ambulation, canalization, resumption of alimentation, and the shorter hospital stay.
cardiac myxoma represents approximately 50% of all benign cardiac tumors in adults, with left atrial myxomas constituting 75% of all myxomas. usually, myxoma is a solitary mass. multicentric cardiac myxomas are very rare and usually, biatrial. the incidence is < 2.5% of all cardiac myxomas. familial multicentric cardiac myxomas may present as a case of left atrial (la) and left ventricular (lv) mass simultaneously. we report a 26-year - old female patient who presented to our emergency department with acute onset of dyspnea and was diagnosed as multicentric right atrium (ra) and right ventricle (rv) myxoma with embolization of tumor fragments to the pulmonary artery (pa) during evaluation with transthoracic echocardiogram and high - resolution computed tomography (hrct) of the thorax. a 26-year - old female presented to the emergency department with sudden onset of dyspnea and left precordial pain of 6 hr duration. she had no history of exertional dyspnea, fever, weight loss, prolonged immobilization, syncope, focal neurological deficits, substance abuse, endocrine disorders and dermatological ailments in the past. the patient 's physical examination revealed blood pressure of 94/60 mmhg, heart rate of 120 beats / min, respiratory rate of 40/min, elevated jugular venous pressure with prominent v wave. on cardiovascular system examination, s1 was normal, s2 was narrowly split with loud p2 ; a pan - systolic murmur of grade iii / vi intensity was present along left sternal border. pulse oximetry revealed a saturation of 85% at room air increasing to 90% with 100% oxygen at 6 l / min. laboratory examination revealed hemoglobin - 12.6 g / dl, total leukocytes count - 12.600/l and platelet count of 1,00,000/l. transthoracic echocardiogram showed a large pedunculated mobile mass in ra with heterogenous echotexture attached to ra free wall and another mass attached to interatrial septum near fossa ovalis [figure 1a ]. another mass was seen in rv measuring 11 mm 11 mm just beneath the septal leaflet of tricuspid valve [figure 1b ]. larger ra mass was prolapsing into rv producing severe eccentric tr jet with a gradient of 60 mmhg [figure 1c, supplementary movie 1 ]. inferior vena cava was free of any mass or thrombus (supplementary movie 2). hrct thorax revealed embolization to the lateral aspect of right pa and along the medial aspect of left pa causing subtotal obstruction with pulmonary infarct in left lung [figure 2a and b ]. (a) modified apical 4 chamber view tte showing a 2.4 cm 2.4 cm pedunculated myxoma in the right atrium (ra) attached to ra free wall extending to interatrial septum, (b) modified apical 4 chamber view tte showing 1.1 cm 1.1 cm pedunculated myxoma attached to the septal leaflet of the tricuspid valve, (c) cw doppler across the tricuspid valve showing severe high - pressure tr with 60 mmhg gradient, (d) tte basal short axis view showing dilated rvot, pulmonary artery and heterogenous pedunculated right atrium mass with calcium specks attached to ra free wall (a) high - resolution computed tomography of the thorax showing embolic fragment in the lateral aspect of right pulmonary artery (pa) and medial aspect of left pa, (b) high - resolution computed tomography thorax showing pulmonary infarct in left lung field once the diagnosis of multicentric ra and rv myxoma with tumor fragment embolization to pa was made, emergency surgery was performed by standard median sternotomy and cardio - pulmonary bypass using bicaval and ascending aortic cannulation to prevent further embolic events. the ra was then opened and a round lobulated mass measuring 3 cm 2.5 cm 2.5 cm was found attached to the ra free wall by a pedicle. another mass attached to interatrial septum near fossa ovalis was excised. multiple degenerating tumor strands were detected in ra, which were not visualized by transthoracic echocardiography. another solitary mass was excised from the rv originating beneath the septal leaflet of tricuspid valve [figure 3a and b ]. after the direct closure of ra, patient was weaned off the extra - corporeal circulation. (a) intra - operative findings showing 3 right atrial myxomas one of which the largest one attached to right atrial free wall, (b) gross morphology of excised myxoma : pedunculated lobulated mass held by forcep and degenerating myxomatous mass with hemorrhage seen on extreme left side of picture, (c) photomicrograph (h and e, 100) loose myxomatoid stroma with stellate and spindle cells (from right atrium mass), (d) photomicrograph (h and e, 45) myxoid stroma with scanty stellate cells and spindle cells amidst degenerating cells and hemorrhage (from right ventricle mass) gross pathological examination revealed multiple flesh - colored soft to firm lobulated mass. gross foci of degeneration were detected. microscopy revealed hypocellular tumor tissue with myxoid background, isolated spindle and stellate cells with no abnormal mitosis or pleomorphism [figure 3c and d ]. gross pathology and histopathological examination were consistent with the diagnosis of cardiac myxoma. after the surgical excision of the tumor, severity of tr decreased from severe to mild. we experienced a case of 26-year - old female with acute pulmonary embolism attributed to embolization of degenerating tumor fragments from multicentric ra and rv myxoma, sporadic in nature, as there was no family history of cardiac tumors in first - degree and second - degree relatives. our patient had no constitutional symptoms of fever, weight loss, malaise or joint pains. pulmonary embolism is the most dreaded and devastating complication of right - sided myxoma. in cases of right atrial myxomas, nevertheless, there have been reports of embolization of thrombi or tumor fragments into the pulmonary vessels in cases of right atrial or right ventricular myxoma. carney 's syndrome consists of myxomas in other locations (breast or skin), spotty pigmentation (lentigines, pigmented nevi or both) and endocrine overactivity (pituitary adenoma, primary pigmented nodular adrenocortical disease, bilateral testicular tumors and schwannomas. familial myxomas tend to occur in younger individuals, are often multiple in locations and more likely to have postoperative recurrences. in the case presented here, despite being a multicentric tumor with younger age at presentation, there was no family history unlike other multicentric cardiac myxomas and thus favoring a sporadic occurrence.. early surgical excision of the cardiac myxoma is advocated to prevent embolization, sudden cardiac death and for relief of obstructive symptoms. cardiac myxomas recur in approximately 1222% of familial cases and in < 3% of sporadic cases. recurrence is attributed to incomplete, inadequate resection of the tumor, multicentricity, younger age at diagnosis and familial myxoma syndrome. annual follow - up examination with echocardiography is recommended in multicentric myxoma postoperatively due to recurrence risk. in patients presenting with sudden onset of dyspnea especially young patients, tumor embolization from right sided tumors should be kept in the differential diagnosis, and timely transthoracic echocardiogram in the emergency room can help in fast - tracking the patient for urgent cardiac surgery to prevent the catastrophic complication of pulmonary embolism and sudden cardiac death.	multicentric cardiac myxoma is a rare syndrome ; usually it is familial. we report a rare case of sporadic right atrium (ra) and right ventricle (rv) myxoma in a 26-year - old female presenting to our hospital for the evaluation of sudden onset of dyspnea and left precordial pain attributed to the embolization of degenerating tumor fragments to the pulmonary artery (pa). the exact incidence of sporadic multicentric ra and rv myxoma presenting as acute pulmonary embolism is unknown as multicentric ra and rv myxoma are very rare. myxomas presenting as pulmonary embolism is < 10%. majority of cardiac myxomas present as exertional dyspnea, chest pain, positional syncope, fever, weight loss and other constitutional symptoms. any young patient presenting with acute onset dyspnea with multiple cardiac masses may have tumor embolization to the pa diagnosis with transthoracic echocardiography and high - resolution computed tomography of thorax, fast - tracks patient transfer for urgent cardiac surgery to prevent further embolization.
the uremic syndrome is attributable to the progressive retention of a large number of compounds called uremic retention solutes or uremic toxins. they include not only small plasma solutes, but also protein - bound solutes and middle molecules (mm)(molecular weight between 500 and 60,000 da). the conventional low flux (lf) dialyzer permits effective small solute clearance, but its clearance of mm is relatively lower. high flux (hf) dialyzer allows more efficient removal of small water - soluble uremic compounds as well as mm and ensures improved dialysis quality and reduces the short- and long - term hemodialysis - related complications. dialysis with hf membranes result in a reduction in erythropoietin resistance, delay in loss of residual renal function, improved lipid profiles, specifically increased high - density lipoproteins cholesterol, lowered triglyceride levels and removal of advanced glycosylation end products, which have been implicated in the pathogenesis of atherosclerosis and dialysis - related amyloidosis. serum creatinine is the most commonly used marker for assessing kidney function in patients with chronic kidney disease (ckd). the use of serum urea is recommended by the kidney disease outcome quality improvement clinical practice guideline to assess dialysis clearance. the urea and creatinine reduction ratios (crr) that are commonly used can assess the removal of only small solutes by conventional hemodialysis. cystatin c is a single nonglycosylated polypeptide chain consisting of 120 amino acid residues with a molecular mass of 13 kd, which is in the mm range. it is produced by all nucleated cells, freely filtered at the glomerulus and virtually fully reabsorbed and metabolized by proximal tubular cells. several studies have suggested that cystatin c is useful as a marker of hemodialysis toxin removal, since it has the attractive features as a representative mm. though hf dialyzers with improved mm clearance are widely used, urea and crr are used to assess the dialysis adequacy. this study aims to assess whether cystatin c reduction ratio (cyscrr) can be used as an alternative indicator of mm clearance in hf hemodialysis. the study was approved by the human ethics committee of sri ramachandra medical college and research institute (srmc), chennai, india, and written consent was obtained from all the participants. a total set of 37 patients of both sexes all the patients were initially subjected to lf hemodialysis and then to hf hemodialysis 2 weeks later. the dialyzers used were f6hps for lf and f60s for hf (fresenius medical care). patients with thyroid dysfunction, malignancies, steroid therapy and hiv infection and pregnant women were excluded from the study. all the blood samples were collected before and after the second hd session of the week, according to the guidelines for hd adequacy. glutamate dehydrogenase method on the biolis premium 24i analyzer manufactured by tokyo boeki medical system, japan. serum creatinine was measured by the modified jaffe 's assay and serum cystatin c was measured by latex - enhanced immunoturbidimetry on the same analyzer. the efficacy of dialysis was then assessed by calculating the reduction ratio for serum creatinine as shown below : crr = 100 (1-ci / co) where ci and co represent post - dialysis and pre - dialysis serum creatinine levels. spss 10 statistical software developed by ibm corporation, united states was used for the analysis of the results. student 's t - test was used for the analysis of the pre- and post - dialysis samples of urea, creatinine and cystatin c. student 's t - test was also used to compare urea, creatinine and cyscrr between lf and hf hemodialysis groups. there is a statistically significant increase in the mean values of cystatin c from the pre - dialysis to the post - dialysis in the lf group [table 1 ]. there is a statistically significant decrease in the mean values of cystatin c from the pre - dialysis to the post - dialysis [table 2 ] in the hf group. urea, creatinine and cystatin c levels in patients undergoing low flux hemodialysis urea, creatinine and cystatin c levels in patients undergoing high flux hemodialysis as shown in figure 1, the difference in the mean values of cyscrr between the lf (9.78 6.705) and the hf (29.27 11.129) dialysis is statistically significant. comparison of urea, creatinine and cystatin c reduction ratios between low flux and high flux hemodialysis krishnamurthy., has shown statistically significant increase in the mean values of cystatin c with a cyscrr of 38% in the lf group. this increase in cystatin c values in the post - dialysis (lf) sample is due to the pore size of the lf membrane (1.5 nm), which does not allow the removal of mm like cystatin c. the electrostatic interaction between the microproteins and other plasma proteins adsorbed onto the dialyzer membrane hinders the filtration of these molecules. cystatin c serves as a surrogate marker of the inadequate clearance of mm by lf membranes. the effective clearance of cystatin c by hf dialyzers is due to the difference in the ultrafiltration rates. cystatin c is removed effectively by the hf membranes as the pore size of the membranes is between 1.5 and 1.7 nm. huang., found no correlation between cyscrr and the small solute clearance (urea reduction ratio and crr). cystatin c, a mm that is distributed mainly extracellularly is minimally protein bound with presumed slow redistribution between the intravascular and the extravascular spaces because of its size. by contrast, serum urea and creatinine are distributed in extracellular (both intravascular and extravascular) and intracellular spaces, with presumed rapid equilibration between all three compartments during hemodialysis. as cystatin c is strictly distributed in extracellular fluid, various kinetic models are not required to describe its kinetics during hd. its production rate is relatively constant or minimally variable ; cystatin c circulates freely in unbounded form and its elimination from the circulation is almost entirely through glomerular filtration. cystatin c has been shown to correlate with mortality in patients with coronary heart disease. in patients with stage iii or iv ckd, it was concluded that if cystatin c levels correlate with clinical outcome in the dialysis population regardless of the residual renal function, it may become an important dialysis adequacy parameter. this study highlights the importance of cystatin c as the dialysis adequacy marker for the clearance of mm in hf dialysis, thus replacing the conventional dialysis adequacy markers of urea and creatinine used in lf dialyzers. further studies with larger sample sizes are required to establish the target of a satisfactory cystatin c level after dialysis that is needed to improve the clinical outcomes in hf dialysis.	the conventional, low flux (lf) dialyzer allows the removal of small molecular solutes like urea and creatinine. high flux (hf) dialyzers allow the effective removal of middle molecules (mm) as well, and are associated with reduced hemodialysis - related morbidity and mortality. cystatin c has attractive characteristics as a representative mm. the aim of this study was to determine cystatin c reduction ratio (cyscrr) in both lf and hf groups and to compare it with other markers of dialysis adequacy. thirty - seven patients were subjected to both lf and hf hemodialysis 2 weeks apart. serum urea, creatinine and cystatin c were measured pre- and post - dialysis. cystatin c was measured by latex - enhanced immunoturbidimetry. urea and creatinine reduction ratios were 72.3 14.7% and 62.5 13%, respectively in the lf group. the cyscrr was 9.7 6.7% and 29.2 11% in lf and hf hemodialysis, respectively. the statistically significant decrease in cyscrr in the hf group shows the effective clearance of mm by hf dialyzers. hence, cyscrr could be applied to measure the mm clearance in hf hemodialysis. this study highlights the significance of cystatin c as an important dialysis adequacy marker replacing the conventional markers such as urea and creatinine in hf hemodialysis. among the middle molecules cystatin c scores over beta-2 microglobulin.
during badminton games, it is necessary for players to use high - level stroke skills in various situations1. players need to be able to select and execute a large number of stroke, in different situations, after selecting highly strategic shots to disrupt their opponent s readiness, rather than simply returning the shuttlecock2. for example, when an opponent takes up the ready position in front of the net, it is appropriate to return the shuttlecock by placing a clear shot behind the opponent, and an opponent takes up the ready position in the back section of the court, a drop shot falling near the net is appropriate. during a rally, a player needs to select the most appropriate shot to disrupt an opponent s readiness, and as soon the opponent s readiness disrupted, deliver the most offensive shot to earn a point3, 4. reports about strokes have been made in some previous studies of badminton games, which focused on the development of skills for delivering effective services and overhead strokes5, 6, or the characteristics of upper - limb muscle activities of skilled badminton players when hitting a smash7. furthermore, in badminton rallies, quick movements are essential for returning the shuttlecocks at various speeds in all directions. to win a rally, increased leg strength enabling rapidly movement to the spots where the shuttlecock falls8, and endurance to continue moving without decreasing the speed of movement9, 10, are crucial. in addition, in order to effectively return a shuttlecock (to disrupt an opponent s readiness), it is necessary to deliver a stroke in a stable stance. in these situations, players need to instantaneously predict the spot where the shuttlecock will fall, and immediately begin to move to it. however, no studies have examined the mechanisms by which badminton players instantaneously react to shuttlecocks, and move. in which demands, instantaneous lower - limb muscle movements are particularly needed. if badminton players have the ability to instantaneously activate their own lower - limb muscles, the excitability of their spinal motor neurons controlling the fibers of such muscles may be characteristic. the soleus h - reflex has frequently been the focus of previous studies11,12,13, as it is considered to represent the excitability of spinal motor neurons. accordingly, in line with this, the present study aimed to clarify the characteristics of badminton players motor neuron excitability by examining the soleus h - reflex in the ready position immediately before making a return. sixteen individuals with experience of playing badminton (mean age : 20.92.1 ; years of experience : 8.33.0) and 16 without such experience (20.11.2) were studied. all subjects were provided with explanations regarding the study objectives and its safety before obtaining their consent to voluntarily participate in this. this study, which was conducted with the approval of the research ethics committee of the health science university (approval number : 13). neuropack (nihon kohden) was used for the measurement of the m- and h - waves. before measurement silver plated electrodes for recording were placed on the skin of the medial part of the soleus on both sides, while bipolar stimulating electrodes were attached to the popliteal fossa of the dominant and non - dominant legs. subsequently, electrical stimulation was intracutaneously applied to the tibial nerves to measure the m- and h - waves in the medial part of the soleus ; and their thresholds and maximum values were recorded by gradually increasing stimulation. for h - wave measurement, the stimulation level was set to obtain h - wave amplitudes of approximately 30% of the maximum m - wave value (mmax), as well as the m - wave of 4 to 8% mmax, at a frequency of 0.2 hz14. when recording the h - reflex, it was confirmed that the m - wave remained unchanged. when changes were observed, the recorded values were discarded due to considering the possibility of changes in the stimulation level. the h - reflex was measured in the ready position before receiving a shuttlecock with or without a racket held in the dominant or non - dominant hand, in addition to a static upright stance. the subjects were instructed to maintain these 5 stances, and the h - wave was recorded 10 times, and the mean was calculated (fig. 1fig. 1.h - wave after tibial nerve stimulation of stance in a representative badminton subject). h - wave after tibial nerve stimulation of stance in a representative badminton subject among the amplitudes obtained for each stance, the difference between the minimum and maximum h - wave values was calculated. subsequently, the difference between the minimum and maximum m - wave values (mmax) as a response to maximum stimulation in the supine position was calculated to normalize the h - wave value as a percentage of the mmax. for statistics, significant differences were examined by performing one - way analysis of variance, followed by multiple comparisons, adopting the tukey - kramer method with a significance level of 5%. the h - wave rate of each stance was calculated, using the values when just standing as 100% (table 1table 1.h - wave in the badminton and control groups in each stancebadmintoncontrol significantholdingdominant (%) 86.510.5108.615.6non - dominant (%) 91.014.7113.527.8without a racketdominant (%) 94.915.2103.316.4non - dominant (%) 90.813.397.815.8 : p<0.05). in the badminton group, the h - wave significantly decreased when holding a racket in the dominant hand compared to when standing. in contrast, in the control group, no significant differences were observed between when standing and the other stances. furthermore, the h - wave was suppressed in all stances compared to when standing in the badminton group, while it was promoted in the control group. muscle stimulation excites type ia afferent fibers, and consequently activates spinal alpha motor neurons, leading to the contraction of stimulated muscles (stretch reflex). the h - wave obtained with electrical stimulation has been used as an index for evaluating the spinal control of muscle contraction during the stretch reflex, as it represents the excitability of spinal motor neurons. in previous studies, the soleus h - wave obtained by eliciting stimuli was shown to be greater when standing compared to walking15, and this may be explained by the mechanism in which the stretch reflex stabilizes the ankle joint when standing (increased h - wave), while it interferes with the swing phase when walking (decreased h - wave). in short, a greater amplitude of the h - wave also varies between different exercise tasks, such as walking and running, and stances, such as prone and standing positions16, 17. furthermore, the soleus h - wave has been reported to be greater in swimmers than in non - swimmers18. on the other hand, the h - reflex level is lower in professional ballet dancers than in athletes in general19. based on these findings, long - term physical training may specifically influence the excitability of spinal motor neurons. in the present study, on comparison of the soleus h - wave of those with and without experience of playing badminton were compared, and its values was significantly lower in the former when experienced players held in the dominant hand, while those with no experience of playng badminton showed a markedly different tendency. some previous studies reported that the h - wave amplitude is greater in athletes mainly engaged in endurance training than in those mainly engaged in power training20,21,22. such training - dependent (endurance / power) variation in the h - wave may be associated with differences in the lower - limb loading level. for example, the h - wave decreases in the prone position compared to when standing under the influence of gravity17, and increases in a microgravity environment or underwater11, 23. in this respect, continuous badminton training suppresses the soleus h - wave, presumably due to being power - focused. in badminton games, it is necessary for players to deal with shots at various speeds, such as drop shots near the net and high - speed smashes, and appropriately the return shuttlecocks. therefore, badminton players need to increase the instantaneous force of their legs to execute rapid movements. the results of the present study suggest that the excitability of badminton players spinal motor neurons may be suppressed by training to increase the instantaneous force (power training). furthermore, the reduced excitability of spinal motor neurons when playing badminton may be associated with an increased ability to move to feet rapidly. considering that badminton is regarded as a lifelong sport for a wide range of age groups, regardless of the sex, and that it increases the stepping ability (by suppressing the excitability of spinal motor neurons), this sport is also likely to be an effective fall - preventing on approach.	[purpose ] this study aimed to clarify the characteristics of motor neuron excitability by examining the soleus h - reflex in the ready position adopted immediately before making a return during badminton games. [subjects ] sixteen individuals with (badminton group) and 16 without (control group) experience of playing badminton were studied. [methods ] each subject was instructed to take up various stances for returning a shuttlecock to measure the h- and m - waves in the soleus. [results ] the h - wave was significantly decreased when gripping a racket was held in the dominant hand than compared to just standing in the badminton group. in contrast, in the control group, no significant differences were observed between when standing and the other stances. [conclusion ] based on these results, the excitability of spinal motor neurons may have been reduced (h - wave suppression) by badminton training to increase the instantaneous force (power training).
the hantavirus cross - sectional survey was carried out april through may 2006 in the municipality of uberlndia, minas gerais, at an average altitude of 863 m (1855s,4816w) (figure). a randomized and stratified (sex and age) sample was collected from the entire rural area and from the south sector of the municipality s periurban area. the term periurban refers to a residential area on the outskirts of the city that is in close contact with the rural area. the participants answered a questionnaire that included demographic information (sex, age, place of birth, and address) and questions relating to hps risk factors (type of dwelling, exposure to rodents at home or work, labor activity, risk activities, history of severe pneumonia, and direct contact with hps patients). blood samples were collected by venipuncture, centrifuged, and sent to the laboratory of hantaviruses and rickettsioses at the oswaldo cruz foundation, rio de janeiro, brazil, for analysis. we screened serum samples by elisa for hantavirus - specific immunoglobulin g using a recombinant antigen of the nucleocapsid protein of araraquara virus, produced in escherichia coli and supplied by the virus research unit of the university of so paulo, brazil, according to the procedure previously described (2). all positive serum samples were retested ; only those that had 3 elisa - positive results at > 1:400 dilutions were considered positive. the mann - whitney u and fisher exact / binomial tests for 2 proportions were applied for comparison among medians and proportions, respectively, using epi info 3.3.2 (www.cdc.gov/epiinfo) and biostat 5.0 (www.biostat.org) software. the 400 study participants comprised 200 rural and 200 periurban residents ranging in age from 12 to 76 years (mean = 41 years). the 8 rural area antibody - positive samples were from male farmers (table 1). presence of antibody was significantly associated with male sex, older age class, and potential risk activities (table 1). although all case - patients reported exposure to rodents or their excreta, this exposure was not statistically significant (table 1). in the periurban area, the presence of antibody was associated with age but not with sex, risk activity, or exposure to rodents (table 1). the mean age of seropositive persons from periurban and rural areas was similar (p = 1.0). the relationship between antibody and sex depended on urban vs. rural residence (p = 0.02). three antibody - positive persons in the rural zone and 2 in the urban zone reported a history of pneumonia, albeit without complications. clearing land, farming, working in pastures or cellars, or cleaning sheds barns, or other outbuildings. the largest number of cases occurred among periurban residents, but the highest cumulative incidence was among rural residents (table 2). nevertheless, rural and periurban areas did not differ significantly in either prevalence or incidence. rural versus periurban. determined by using 2-tailed fisher exact or binomial tests for 2 proportions. overall hantavirus antibody prevalence among periurban residents was 2.0%, with a higher prevalence among women (2.6%). in previous studies, the prevalence of hantavirus antibodies was higher in men (46). this finding is similar to a situation reported in colombia, where all positive samples came from men engaged in rural activities (6). these activities involve a high risk for infection by hantaviruses (7). in this study, hantavirus positivity was found only in persons > 39 years of age, and the difference in the mean age of the participants in relation to positivity was significant. this fact might suggest a historic high - risk event to which the older age class, but not the younger age class, was exposed. high hantavirus antibody prevalence has been found in studies of some human populations in latin america (5,8,9). the prevalence of araraquara virus reactive antibodies among the volunteers in this study demonstrates that transmission is not rare, reinforcing the hypothesis of the existence of mild disease or asymptomatic infections (10). two hypotheses have been proposed : clinically mild disease or inapparent infections might result from differences in the nature of exposure (e.g., low inoculum or inefficient mechanism of transmission) or genetic differences in immune response to infection, or they might indicate the circulation of > 1 hantavirus genotypes of greatly reduced virulence (10,11).	a cross - sectional serosurvey was conducted to assess the proportion of persons exposed to hantaviruses in a virus - endemic area of the state of minas gerais, brazil. findings of this study suggested the presence of > 1 hantaviruses circulating in this region causing hantavirus pulmonary syndrome, mild disease, or asymptomatic infection.
most coronary artery origin abnormalities are incidentally determined during coronary angiography. in the adult population, its prevalence is reported to be approximately 0.3 to 1.3% in the largest registry.1) these abnormalities are usually asymptomatic and have no clinical significance. however, some cases of coronary artery abnormalities are related to severe life - threatening events such as myocardial ischemia, arrhythmia and acute myocardial infarction.2) we report an uncommon case of an anomalous origin of the left coronary artery (lca), a single coronary artery, arising from the right sinus, with angina pectoris and palpitations. a 48-year - old woman presented with exertional angina and palpitations for a long time. she a had medical history of hypertension and dyslipidemia, and a family history of coronary artery disease. the exercise electrocardiogram showed dynamic changes with st - segment depression in the v 1 - 4 leads. the coronary angiography procedure was started with a lca cannulation attempt, but left anterior descending coronary artery and cx imaging was unsuccessful even though contrast was injected into the left coronary sinus. the right coronary artery was cannulated and visualized with a right judkins catheter. at this time the right judkins catheter was gently pull back and the left coronary arteries were clearly visualized (figs. 1 and 2). coronary ct angiography confirmed that the left coronary arteries arose from the right sinus of valsalva and that all three coronary arteries originated from the single sinus (fig. the patient was managed with conservative treatment and has had no symptoms on clinical follow - up. coronary anomalies affect less than 1% of the general population. anomalous origin of lca from the right sinus of valsalva is the rarest anomaly, with a reported prevalence of 0.02 - 0.03% according to studies. the isolated origin of a single coronary artery is very rare, with an incidence of 0.04% to 0.23%.3) most of these coronary artery anomalies are generally asymptomatic ; however, some can cause severe potentially life threatening events. understanding anatomic coronary variations is important in determining anomalous origins that are related to sudden cardiac death.4) single coronary artery has been defined angiographically by lipton.5) according to the origin from the coronary artery. the modified lipton classification includes features such as the anatomical distribution, the ostial location, and the course of the transverse trunk. anomalous origin of the lca from the right sinus of valsalva is associated with sudden death in some cases (59%) because an anomalous artery between two great vessels is related with acute myocardial infarction and sudden cardiac death.6) the acute angle of the ostium increases the risk of sudden cardiac death. the anomaly determined in our patient seemed to be potentially malignant, but without marked compression between the great arteries. the management of patients with an anomalous origin of coronary artery includes observation, medical treatment, coronary stent implantation and surgery repairment. however, in our case medical treatment was chosen due to the absence of compression of the coronary arteries by the great arteries, and the lack of acute ostial angulation. the patient was treated with a beta blocker and nitroglycerin because of the angina pectoris and palpitations, and she has remained asymptomatic for 1 year on follow - up. although cardiac catheterization is generally accepted as the gold standard for the evaluation of coronary anomalies, ct angiography has recently emerged as an effective and noninvasive method for performing imaging of the origin of the coronary arteries. in conclusion, the determination of the anomalous origin of the coronary artery and the cardiovascular system is of great clinical importance due to its severe life - threatening complications. the new imaging modalities that have emerged enable the accurate visualization of the anatomical configurations and the detection of structural malformations. most of the structural cardiovascular abnormalities are incidentally detected and are asymptomatic ; however, a few are potentially significant and can trigger sudden death.	anomalous origin of coronary arteries is generally asymptomatic and a rare disease. however, some cases can cause severe life - threatening events such as myocardial ischemia, arrhythmia, and acute myocardial infarction. we describe a case of a single coronary artery arising from the right sinus of valsalva in a 48-year - old female patient with a complaint of stable angina pectoris and palpitations. coronary angiography revealed that all three coronary arteries arose from the right sinus. coronary ct angiography confirmed that there was an anomalous origin of the left coronary artery arising from the right sinus of valsalva. the patient was managed with conservative treatment.
the line listing of ha patients included persons with laboratory evidence of recent hav infection (anti - hav immunoglobulin m [igm ]) who stayed at hotel x after june 1, 2004. also listed were hotel guests with ha disease (jaundice, elevated liver enzyme levels), without laboratory confirmation, who had traveled with persons with laboratory - confirmed cases. a case - control study was performed among hotel x guests > 17 years of age residing in 3 german states. the time span between the earliest arriving case - patient 's last day at hotel x and the latest arriving case - patient 's first day there was defined as the " minimum period of transmission " (mpt). healthy hotel guests who stayed at the hotel during the mpt who had neither been vaccinated against hav nor previously infected with hav were eligible as controls. telephone interviews were conducted with a standardized questionnaire that elicited information on demographic factors, foods and drink consumption, and participation in recreational activities such as swimming, day trips, etc. for statistical analysis, exposures were dichotomized into " ever " versus " never, " and the " number of days exposed " was calculated. univariate analysis (tests) and multiple logistic regression were performed (spss version 12.0.2, spss inc., chicago, il, usa) ; the egyptian authorities ' investigation included testing all hotel employees for hav antibodies (igm, igg), and scrutiny of food suppliers. serum samples were obtained from german case - patients for testing by reverse transcription pcr (rt - pcr) in the vp12a junction and sequencing of a 160-bp - long pcr fragment of the vp1 region. the outbreak lasted from july 10 to september 8, 2004 (figure 1). a total of 351 case - patients made up this outbreak : 271 primary and 7 secondary infections in germany, plus 60 primary infections reported to the national public health institutes in 8 other european countries. austria recorded a secondary outbreak with 13 cases caused by an infected food handler who had stayed at hotel x (3). in preceding years, in germany only 28 ha cases were reported after travel to egypt with disease onset between july 10 and september 8. epidemic curve : distribution of dates of disease onset for outbreak - associated hepatitis a case - patients from germany (n = 264), and minimum period during which hepatitis a virus transmission occurred (mtp). of the german reported primary cases, overt clinical ha developed in 263 (97%) persons. case - patients were 2 to 67 years of age (median 34 years) and 54% were male. no more than 52% of the case - patients had stayed together at the hotel on any single day. case - patients had stayed at hotel x from 6 to 21 days, and 70% had stayed 13 days or longer. sixty - nine ha case - patients (60% response among the 115 case - patients in the 3 states) and 36 controls were included in the statistical analysis. eighty - seven percent of the case - patients reported absence from work for 3 to 56 days (median 26 days), and 54% of the case - patients were hospitalized for 2 to 25 days (median 9 days). case - patients and controls did not differ significantly by age, sex, recreational activities, consumption of ice cream or salads, or other foods consumed or behavioral characteristics. case - patients were significantly more likely to have drunk orange juice served at the breakfast buffet (82%) than were controls (64%) (odds ratio 2.6 ; 95% confidence interval 1.16.6). a dose - response relationship became apparent between number of days of orange juice consumption and ha (figure 2). case - patients consumed orange juice for a median of 11 days, and controls consumed it for a median of 5 days. in 22 (52%) of the 42 serum samples available for testing, all samples compared by sequencing were identical and belonged to genotype 1b (figure 3). days of orange juice consumption among hepatitis a patients and controls. or, odds ratio ; ci, confidence interval. phylogenetic analysis of the vp12a junction region. the sequence of a 160-bp - long pcr fragment (isolate egy/1/05/deu, genbank accession no. ay741663) of the hepatitis a vp1 region was compared to published reference sequences of hepatis a virus. aj306374(arg-1), ay656712 (bav/2/03/deu), ay028976 (sa/10/2000/deu), ay046073 (datteln/01/deu), and ay747173 (nrw/3/04/deu). genotype ib : l07728 (jor 88), m14707 (hm-175). the scale represents nucleotide substitutions per position (denotes previous german outbreak - causing strains). the on - site investigations in egypt did not identify hotel staff positive for hav igm. minimal fluctuation among hotel staff renders it unlikely that an hav - positive employee was missed. this large outbreak demonstrates risk and clinical impact of ha for nonimmune travelers to ha - endemic countries. in germany, the outbreak accounted for 12% of all ha case - patients notified in the year 2004. the results of the outbreak investigation strongly point to orange juice as the infection vehicle. in the case - control study, among a broad range of foodstuff, beverages, and recreational activities queried, the consumption of orange juice was the only exposure significantly associated with ha, with higher doses of juice significantly increasing ha risk. these findings are corroborated by the inspection of the hygienic conditions under which the juice was produced in egypt. the juice was most likely contaminated during the manufacturing process, e.g., by an infected worker with imperfect hand hygiene or by contact of fruit or machinery with sewage - contaminated water. citrus fruit and citrus juices have only rarely been implicated as vehicles of ha outbreaks, with contamination typically described during preparation just before consumption (4,5). salmonella outbreaks caused by orange juice contaminated at the production site have been identified (68). as hav is quite resistant to acid (9), it likely survives for prolonged periods in orange juice. less stable pathogens such as escherichia coli have been shown to survive in orange juice for > 15 days (10). the fact that juice was consumed by 60% of healthy controls may be explained in part by fluctuating virus concentration within the juice, which resulted in varying degrees of infectiousness during the 4-week period. a contaminated lot may have been phased - in and out slowly by gradual mixture with other lots. also, the study design did not allow the exclusion of controls who did not know they were immune. the hurghada outbreak - strain clearly differs from strains that have caused nontravel - associated outbreaks in germany in recent years. two large autochthonous outbreaks were caused by hav type 1a strains (11). in the netherlands, hav strains in autochthonous cases mostly also belonged to hav 1a, whereas 1b strains were found more often in children of moroccan origin (12). extended monitoring of hav strains, for example, as performed in the united states (13), could find hidden clusters and demonstrate links between imported and autochthonous cases. vaccination against ha is recommended for all nonimmune travelers to ha - endemic areas (14). this outbreak showed that a high proportion of german tourists in egypt were either not adequately informed about ha risk and the benefits of vaccination or were informed yet still decided against vaccination. the outbreak emphasizes the importance of adequate pretravel advice, preferably from an institution specialized in travel medicine.	in 2004, a major outbreak of hepatitis a among tourists returning from egypt involved 351 case - patients from 9 european countries who were infected with a single strain (genotype 1b). the case - control study identified orange juice as the most likely infection vehicle. vaccination against hepatitis a virus is strongly recommended before travel to disease - endemic areas.
a 61-year - old man complaining of syncope and chest pain came to our hospital. ten days prior to hospitalization, he had had a fever, chills, and body ache. he had taken some medication from a local clinic for these symptoms, which were supposedly caused by a simple upper respiratory infection. despite the medication, the patient had developed another symptom of syncope 7 days earlier and was transferred to the neurology department for evaluation. on arrival, he had mild fever (37.5c) with chills, sweating, and dyspnea. the blood pressure was 110/40 mmhg, pulse rate 90/min, and respiratory rate 20/min. on the chest auscultation, the chest x - ray revealed cardiomegaly, and the electrocardiogram demonstrated a complete av block. in the brain magnetic resonance imaging, there were multiple infarcts, which were thought to be caused by septic emboli. cardiopulmonary bypass was initiated, and the ascending aorta was then cross - clamped. after infusing the histidine - tryptophan - ketoglutarate solution through the retrograde cardioplegic catheter and local cooling with ice slush, transverse aortotomy was performed. the aortic valve was thickened and retracted ; this was consistent with a rheumatic aortic stenosis. there was a commissural fusion between the noncoronary cusp and the right coronary cusp, which also had fibrous thickening with calcification and abscess (fig. 1). the periannular abscess involved the aortic root of the base of noncoronary sinus (fig. 2) and invaded down to the central fibrous body, whole membranous septum, and mitral and tricuspid valves (tvs) (fig. the aortic valve was excised carefully, and then, the infected tissue and the pus were removed meticulously from the abscess pocket down to the membranous septum, central fibrous body, annulus of the mitral valve (mv), and anteroseptal commissure of the tv. we also made an extended transseptal incision extending from the right atrial auricle to the tv to expose the lesion and achieve complete debridement. the septal and anterior leaflets of the tv were also partially resected due to vegetations. after the complete debridement of the infected area, we found a large defect including the left ventricular outflow tract (lvot), which contained the membranous septum, central fibrous body extending to the tv, and mitral annulus. the reconstruction of the lvot was performed with half - folded elliptical bovine pericardium (peri - guard ; synovis surgical innovation, st. the bovine pericardium was trimmed into an elliptical shape and a half - fold was made ; then, its base was secured by interrupted suturing to the annulus of the mv by using reinforcement with a teflon felt, followed by lvot reconstruction using a leaflet of the half - folded elliptical bovine patch with aortic valve implantation concomitantly using a 21-mm mechanical valve (sjm masters series valve ; st. 3). after closing the aortotomy site, tricuspid annuloplasty was done : partial excision and re - suspension of the septal and the anterior leaflet and anteroseptal commissuroplasty. the other half of the half - folded elliptical bovine patch was used for repairing the right atrial wall with a defect and tv. after weaning the patient from cardiopulmonary bypass, we conducted transesophageal echocardiography, which revealed neither an intracardiac shunt nor tv and mv regurgitation. because the complete av block was observed, we began temporary pacing at the rate of 80 beats / min after placing permanent pacing leads on the right atrial and ventricular walls. the total cardiopulmonary bypass and aortic cross clamping times were 303 minutes and 238 minutes, respectively. on postoperative day 1, we were able to wean the patient from mechanical ventilation and extubate an endotracheal tube. intravenous antibiotics with ampicillin, nafcillin, and gentamicin were continued for approximately 6 more weeks. the echocardiography on postoperative day 51 revealed normal lv contractility, no intracardiac shunt, and a well - functioning prosthetic mechanical aortic valve without para - valvular leakage and tricuspid regurgitation. on postoperative day 56 infective endocarditis with abscess formation is a rare, life - threatening condition, and it exhibits a high postoperative mortality. this medical condition is fatal because of the complicated anatomical features of the fibrous skeleton. this is also particularly true when the fibrous skeleton of the heart is extensively destroyed and the reconstructive procedures are complicated. the fibrous skeleton of the heart is a complex framework that surrounds the orifices of the valves, the right and left fibrous trigone, and the membranous parts of the interatrial and interventricular septa. our case displayed fibrous skeleton destruction, which extended even to the mv and tv. after the meticulous debridement and resection of all infected and necrotic tissues, an appropriate reconstruction of defects ensured better surgical results. unfortunately, current literature regarding the surgical management of fibrous skeleton destruction is limited. in the presence of endocarditis, annular destruction with a loss of ventricular or mitral aortic continuity david. described a surgical method that involves replacing aortic and mvs after the reconstruction of the intervalvular fibrous body by using two triangular patches of glutaraldehyde - fixed bovine pericardium. they insisted that the glutaraldehyde - treated bovine pericardium is an excellent material for use in reconstructing the heart in patients with paravalvular abscess. presented a case of a combination of annuloaortic ectasia and infectious endocarditis that required reconstruction of the aortic root and aorto - mitral common annulus, and mitral valve replacement. black. reported that konno aorto - ventriculoplasty, originally described for the enlargement of the aortic annulus and the lvot, may be well suited for the debridement and repair of fibrous skeleton endocarditis. we present a case of the destructed fibrous skeleton including lvot, right fibrous trigone, membranous septum, and tricuspid and mitral annulus which we successfully reconstructed with a bovine pericardial patch that had a half - folded elliptical shape. one leaflet of this patch was applied for lvot reconstruction from the mv with aortic valve implantation, and the other for the right atrial wall with a defect and tv. in conclusion, in the reconstruction of the lvot with a defect, right fibrous trigone, and tv in the infective endocarditis, one half - folded elliptical pericardial patch could be a useful option to repair both the lvot and the right - side area with a defect.	a 61-year - old man was diagnosed with aortic stenoinsufficiency with periannular abscess, which involved the aortic root of noncoronary sinus (ncs) that invaded down to the central fibrous body, whole membranous septum, mitral valve (mv), and tricuspid valve (tv). the open complete debridement was executed from the aortic annulus at ncs down to the central fibrous body and annulus of the mv and the tv, followed by the left ventricular outflow tract reconstruction with implantation of a mechanical aortic valve by using a leaflet of the half - folded elliptical bovine pericardial patch. another leaflet of this patch was used for the repair of the right atrial wall with a defect and the tv.
the ectodermal dysplasia represents a group of inherited conditions in which two or more ectodermally derived anatomic structures fail to develop. ectodermal dysplasias represent a large and complex group of diseases comprising of more than 170 clinical conditions. depending upon the presence or absence of sweat glands, it is divided into the hidrotic (clouston syndrome) and anhidrotic types. (cst - syndrome i.e. christ- siemen - touraine syndrome and anhidrotic / hypohidrotic, ectodermal dysplasia being synonymous.) in most cases, this disorder seems to show an x - linked pattern, with gene mapping to xq12-q13.1 ; therefore, a male predominance is usually seen. individuals affected by it show the triad comprising anhydrosis / hypohydrosis, hypotrichosis, and dental hypoplasia. hypodontia has been considered to be a multifactorial condition with genetic and environmental influences, and published opinions differ on the importance of each factor. larmour., state that recent developments in molecular genetics are revealing the roles of the homeobox genes in the control of the complex epithelial / mesenchymal interactions that occur during dental development. those of particular interest for dental development are the muscle - specific homeobox genes, msx1 and msx2. here is the case report of a 8-year - old male child with ectodermal dysplasia. the chief complaint of the patient was difficulty in mastication due to absence of maxillary and mandibular teeth. the patient experienced episodes of high fever, was intolerant to heat, and did not sweat. his i.q., level was low, and it was evident when asked about schooling. other characteristics of ectodermal dysplasia, such as frontal bossing, saddle nose, reduced vertical dimension of face due to total anodontia were also noticed. child showing dry skin, sparse eyebrows, eyelashes, and scalp hair hands showing dystrophic (thin and brittle) nails feet showing dry skin and dystrophic nails intraoral examination revealed absence of teeth with thin alveolar crests [figures 4 and 5 ]. complete anodontia of maxillary arch complete anodontia of mandibular arch occlusal and panoramic radiographs revealed no primary and permanent teeth [figures 68 ]. in order to improve mastication and aesthetics, both upper and lower complete dentures were fabricated [figure 9 ] occlusal view of maxilla occlusal view of mandible orthopantomogram (opg) showing complete anodontia post treatment photograph ectodermal dysplasia is one of the most important anomalies of interest to dental clinicians because of the absent or misshapen teeth. hypodontia is known as one of the major factors of ectodermal dysplasia and is almost always present. in severe cases, no teeth form. the absence of primary teeth (true anodontia) is a rare phenomenon. in this case, the patient 's history and clinical and radiographic examination revealed the absence of primary teeth. acikgoz., and vieira., also reported true anodontia of primary teeth. total anodontia denoted by complete developmental absence of teeth in both primary and secondary dentitions was reported pirgon. and pannu and singh. it is claimed that primary teeth must be present for the development of their permanent successors. there are no permanent teeth in the oral cavity of the patient, similar finding reported by vieira. the patient experienced episodes of high fever, was intolerant to heat, and did not sweat. child 's inability to perspire, more comfortable during cold weather, and absence of hair from the eyebrows with scanty eyelashes were also reported by gupta. and pannu and singh (2002). short stature, underweight in relation to age and mental retardation were reported in accordance to case reported by gupta. management : to improve the appearance, mastication, and speech, the child was provided with maxillary and mandibular complete dentures similar to treatment provided by vierra. the dental team should be aware of the clinical presentation of ectodermal dysplasia in order to provide the correct guidance for functional, social, and psychological needs of the patients.	the hereditary condition known as ectodermal dysplasia is characterized by the absence or defect of two or more ectodermally derived structures. the most commonly observed forms of ectodermal dysplasia are the hidrotic and hypohidrotic types ; discrimination is based on the absence or presence of sweat glands. a case of 8-year - old male child with hypohidrotic ectodermal dysplasia with complete anodontia of primary as well as secondary dentitions is presented. the child had a short stature, low intelligent quotient (i.q.,), and was underweight. the patient experienced episodes of high fever, was intolerant to heat, and did not sweat. he exhibited smooth and dry skin, sparse light - colored eyebrows. dental clinicians can be the first to diagnose ectodermal dysplasia due to the absence of teeth.
mutations were introduced into has by megaprimer mutagenesis (sarkar and sommer 1990 ; thomas and roth, 1994). the pcr product was digested with xbai and bamhi and used to replace an xbai to bamhi fragment in an expression vector pcb6-ha - y543 xba that encoded the 133 carboxyl - terminal amino acids of japan ha. each pcr fragment was sequenced to confirm the identity of the mutation and that second - site mutations had not occurred. mdck subline d5 cells (brewer and roth, 1995) were transfected with ha mutants in the expression vector pcb6 (brewer, 1994) and selected for survival in g418. the uncloned, drug- resistant cell population was used for experiments to avoid any effects from clonal variation. mdck cell monolayers were grown, treated with 10 mm sodium butyrate, and then labeled with 1.14 ci / ml transslabel (icn biomedicals, inc., irvine, ca) as described (lin., 1997). to measure rates of entry to the golgi complex, monolayers expressing each mutant ha that had been labeled with radioactive amino acids were chased with prewarmed nonradioactive medium at 37c for either 0, 10, 20, 30, or 60 min. the immunoprecipitates were analyzed by page and quantified by scanning with a phosphorimager (molecular dynamics, inc., the proportion of each immunoprecipitated protein that had shifted to the slower migrating, golgi - modified form was measured as a fraction of the total labeled ha in the immunoprecipitate. to measure rates of arrival at the cell surface, cells were pulse - labeled as described above and chased at 37c in dme containing 10 g / ml of trypsin for 0, 30, 60, 120, or 180 min. trypsin cleaves ha arriving at the cell surface into two disulfide - bonded fragments, ha1 and ha2. the cells were shifted to ice and treated with 100g / ml of soybean trypsin inhibitor (sti) for 15 min, and then has were immunoprecipitated, separated by page, and the amount of ha0, ha1, and ha2 was quantified. for each sample, the percentage of ha at the cell surface was calculated as ([ha1 + ha2]/ total ha) 100%. for the 4a511 protein, the chase medium did not contain trypsin and arrival at the cell surface was measured by biotinylation at 4c as previously described (brewer and roth, 1991). these assays were essentially as previously described (skibbens., 1989 ; lin., 1997). for assays of polarized transport, confluent mdck monolayers expressing each mutant ha were grown for 5 d on filter culture inserts. cells were treated with dme containing 10 mm sodium butyrate for 15 h to increase ha expression and were pulse labeled as described (lin., 1997). for experiments in which cholesterol was depleted from the cells before the chase, cells were grown for 4 d in dme containing 10% fetal bovine serum. 16 h before the chase, cells were treated with dme containing 10% lipoprotein - deficient newborn calf serum and 50 m of compactin (hua., 1996). 50 m of compactin was also present in media used for labeling with radioactive amino acids. after pulse labeling, has were chased to the cell surface in serum - free dme. for each ha, one sample had trypsin present in the apical chase medium, one had trypsin in the basolateral chase medium, and one had no trypsin. excess sti was always included in the medium on the side of the filter opposite to that containing trypsin. at the end of the chase, samples were treated with sti and the has were immunoprecipitated and analyzed as described for the cell surface assay. the percentage of ha at the apical surface was calculated by dividing the fraction of labeled ha that was cleaved by trypsin at the apical surface by the fraction of labeled ha that was cleaved at both apical and basolateral surfaces (the surface population) 100%. each value was corrected for the fraction of ha1 + ha2 in samples chased in the absence of trypsin, which was usually < 10%. for assays of insolubility in triton x-100 cells were then treated for 15 h with sodium butyrate, were labeled with 1.14 ci / ml transslabel for 15 min at 37c, and then chased in normal medium for 80 min. cells were extracted on ice for 30 min in 50 mm tris - hcl, ph 7.5, containing 150 mm nacl, 2 mm edta, 2 mm dtt, 1 protein inhibitor set (boehringer mannheim biochemicals, inc., indianapolis, in), 100 g / ml sti, and 1% triton x-100. cells were scraped into 1.5-ml microfuge tubes and were centrifuged at 12,000 g for 5 min (model microfuge r ; beckman coulter, fullerton, ca). pellets were solubilized with 50 mm tris - hcl, ph 8.8, containing 5 mm edta and 1% sds and passed several times through a 27-gauge needle. the pellet fraction was diluted with extraction buffer and supernatant fractions with sds so that the final concentration of sds in each was 0.17%. mutations were introduced into has by megaprimer mutagenesis (sarkar and sommer 1990 ; thomas and roth, 1994). the pcr product was digested with xbai and bamhi and used to replace an xbai to bamhi fragment in an expression vector pcb6-ha - y543 xba that encoded the 133 carboxyl - terminal amino acids of japan ha. each pcr fragment was sequenced to confirm the identity of the mutation and that second - site mutations had not occurred. mdck subline d5 cells (brewer and roth, 1995) were transfected with ha mutants in the expression vector pcb6 (brewer, 1994) and selected for survival in g418. the uncloned, drug- resistant cell population was used for experiments to avoid any effects from clonal variation. mdck cell monolayers were grown, treated with 10 mm sodium butyrate, and then labeled with 1.14 ci / ml transslabel (icn biomedicals, inc., irvine, ca) as described (lin., 1997). to measure rates of entry to the golgi complex, monolayers expressing each mutant ha that had been labeled with radioactive amino acids were chased with prewarmed nonradioactive medium at 37c for either 0, 10, 20, 30, or 60 min. the immunoprecipitates were analyzed by page and quantified by scanning with a phosphorimager (molecular dynamics, inc., the proportion of each immunoprecipitated protein that had shifted to the slower migrating, golgi - modified form was measured as a fraction of the total labeled ha in the immunoprecipitate. to measure rates of arrival at the cell surface, cells were pulse - labeled as described above and chased at 37c in dme containing 10 g / ml of trypsin for 0, 30, 60, 120, or 180 min. trypsin cleaves ha arriving at the cell surface into two disulfide - bonded fragments, ha1 and ha2. the cells were shifted to ice and treated with 100g / ml of soybean trypsin inhibitor (sti) for 15 min, and then has were immunoprecipitated, separated by page, and the amount of ha0, ha1, and ha2 was quantified. for each sample, the percentage of ha at the cell surface was calculated as ([ha1 + ha2]/ total ha) 100%. for the 4a511 protein, the chase medium did not contain trypsin and arrival at the cell surface was measured by biotinylation at 4c as previously described (brewer and roth, 1991). these assays were essentially as previously described (skibbens., 1989 ; lin., 1997). for assays of polarized transport, confluent mdck monolayers expressing each mutant ha cells were treated with dme containing 10 mm sodium butyrate for 15 h to increase ha expression and were pulse labeled as described (lin., 1997). for experiments in which cholesterol was depleted from the cells before the chase, cells were grown for 4 d in dme containing 10% fetal bovine serum. 16 h before the chase, cells were treated with dme containing 10% lipoprotein - deficient newborn calf serum and 50 m of compactin (hua., 1996). 50 m of compactin was also present in media used for labeling with radioactive amino acids. after pulse labeling, has were chased to the cell surface in serum - free dme. for each ha, one sample had trypsin present in the apical chase medium, one had trypsin in the basolateral chase medium, and one had no trypsin. excess sti was always included in the medium on the side of the filter opposite to that containing trypsin. at the end of the chase, samples were treated with sti and the has were immunoprecipitated and analyzed as described for the cell surface assay. the percentage of ha at the apical surface was calculated by dividing the fraction of labeled ha that was cleaved by trypsin at the apical surface by the fraction of labeled ha that was cleaved at both apical and basolateral surfaces (the surface population) 100%. each value was corrected for the fraction of ha1 + ha2 in samples chased in the absence of trypsin, which was usually < 10%. for assays of insolubility in triton x-100 cells were then treated for 15 h with sodium butyrate, were labeled with 1.14 ci / ml transslabel for 15 min at 37c, and then chased in normal medium for 80 min. cells were extracted on ice for 30 min in 50 mm tris - hcl, ph 7.5, containing 150 mm nacl, 2 mm edta, 2 mm dtt, 1 protein inhibitor set (boehringer mannheim biochemicals, inc., indianapolis, in), 100 g / ml sti, and 1% triton x-100. cells were scraped into 1.5-ml microfuge tubes and were centrifuged at 12,000 g for 5 min (model microfuge r ; beckman coulter, fullerton, ca). pellets were solubilized with 50 mm tris - hcl, ph 8.8, containing 5 mm edta and 1% sds and passed several times through a 27-gauge needle. the pellet fraction was diluted with extraction buffer and supernatant fractions with sds so that the final concentration of sds in each was 0.17%. to investigate a possible role for the ha tm in apical transport of ha in mdck cells, a cdna for ha was mutated to produce a series of has in which blocks of four or five contiguous amino acids were converted to alanines at different positions throughout the entire tm (fig. 1). in addition, since it has been proposed that interactions between lipid head groups might stabilize the association of lipids in digs (simons and ikonen, 1997), four amino acids preceding the tm were also changed to alanine. a highly polarized mdck clonal cell line, d5, used previously by us (thomas and roth, 1994 ; lin., 1997), was transfected with expression plasmids containing the mutant cdnas and uncloned, polarized mdck cell populations were selected that stably expressed mutant has. since the ha tm can influence the folding and trimerization of the protein (lazarovits., 1990), and trimerization is required for efficient export of ha from the er (copeland., 1986 ; gething., 1986), the rate at which each protein reached the golgi complex or cell surface was measured by pulse - chase experiments (table i). only one of the mutant proteins, ha 2a514, (fig. 1), was found to be severely defective in transport to the golgi complex. the other proteins were exported from the er to the golgi complex at rates that ranged from 50 to 100% of the rate of the wild - type ha. properly folded ha is cleaved by extracellular trypsin at a single site, producing mature, biologically active ha composed of disulfide - linked ha1 and ha2 subunits (wiley and skehel, 1987). when pulse - labeled mutant has were chased in medium containing trypsin, only ha 4a511 was degraded by the trypsin, indicating that it was not properly folded. the other mutant proteins were cleaved into ha1 and ha2 and reached the cell surface as fast or faster than wild - type ha, and to a comparable extent, with the exception of ha 2a514 which was retained in the er. mdck cells expressing each of the ha mutants or the wild - type ha were grown as confluent monolayers on filter culture inserts, and the ability of the cells to sort ha into the apical pathway was determined by pulse - chase protocols (matlin and simons, 1984 ; brewer and roth, 1991). an image of a typical experiment is shown in fig. 2 a. we estimate from labeling experiments such as this that expression of the various mutant has differed by no more than fourfold and in each case was relatively low, at least 50-fold less than in mdck cells infected with influenza virus (data not shown). no correlation between sorting of has and expression level in these cell lines was observed. the results of many experiments measuring delivery of has to the apical and basolateral surfaces of mdck monolayers grown on filter inserts are presented in table ii. changing the last five amino acids of the ha tm sequence to alanines (5a531) changing sequences of four or five amino acids to alanines on the other side of the transmembrane domain, either before (4a507) or immediately after the point where the chain is predicted to enter the outer bilayer (2a511, 4a511) caused the protein to be delivered equally to the apical and basolateral surface. however, mutation of residues predicted to lie near the base of the outer leaflet of the bilayer had a much more dramatic effect. this is seen most clearly with the 2a520 protein, which was sorted more efficiently to the basolateral surface than the wild - type ha was sorted to the apical. thus, there is a critically important region near the center of the ha tm that is necessary for the protein to enter into apical transport pathways (table ii) and mutation of this region did not affect the folding of the external domain of ha (table i). mutation of residues immediately beyond position 520, which would be predicted to lie within the inner leaflet of the bilayer, also prevented apical sorting but were less severe. to investigate the individual contributions for apical sorting of residues g520 and s521 of the a / japan/305 ha, each was mutated in separate cdnas. mutation of s521 to alanine produced as severe a defect as deleting both g520 and s521, and caused the s521a protein to be delivered predominately to the basolateral surface. scheiffele and colleagues (1997) have shown that the ability of the ha to associate stably with digs in fibroblasts is sensitive to changes in the sequences of the portion of the tm that span the outer leaflet of the bilayer. the sequences that would span the inner leaflet were found to be relatively unimportant for this property. we confirmed these observations in mdck cells expressing the ha tm mutants (figure 2 b and table ii). consistent with the hypothesis that association with detergent - insoluble membrane domains is required for proper sorting of ha into an apical transport pathway, we did not observe proper sorting of any mutant protein that was soluble in triton x-100. however, mutants 5a528, a516s, and g520s were as insoluble in triton x-100 as was wild - type ha, but were not sorted properly to the apical surface. thus, association of ha with digs appears to be necessary, but not sufficient, for apical sorting. the observation that the 2a520 mutant was transported predominately to the basolateral surface of mdck cells could be explained either by exclusion of most of the mutant protein from apical transport pathways, forcing it into the basolateral pathway, or by creation of a positive basolateral signal that allowed active basolateral sorting. there has been no previous evidence for either a cryptic basolateral sorting signal in ha or for the existence of basolateral sorting signals in tm domains. however, there is evidence that efficient basolateral sorting of the na / k atpase requires an intact apical pathway containing digs, which presumably excludes the na / k atpase and forces it into the basolateral pathway (mays., 1995). if basolateral sorting of the 2a520 mutant occurred by a similar mechanism, then treatments that inhibited the formation of digs might allow ha 2a520 access into apically directed vesicles, and the protein should be exported randomly. digs require cholesterol to form (schroeder., 1994 ; scheiffele., 1997). thus, mdck cells expressing either the ha 2a520 mutant, wild - type ha, or ha+8 d549h, which contains a dominant basolateral signal (lin., 1997), were grown as monolayers and depleted of cholesterol. under the conditions used, the ability of ha to partition into membranes insoluble in triton x-100 was decreased by 50%. the delivery of each of these has to the plasma membranes of cells depleted in cholesterol was measured (table iii). depletion of cholesterol had no effect on the apical transport of wild - type ha, indicating that the apical pathway functioned under these conditions. however, twice as much ha 2a520 reached the apical surface in cholesterol - depleted cells as did when cholesterol levels were normal, resulting in randomized transport of ha 2a520. this suggests that the presence of digs in cells having normal levels of cholesterol excluded this mutant from apical pathways. cholesterol depletion had no effect on the basolateral pathway, as shown by continued basolateral transport of ha+8 d549h. the effects of mutations in the transmembrane domain of the a / japan ha are summarized in fig. 3. residues i514 and y515 were critical for proper folding of this ha. although there was some reduction in transport rates observed for mutants with changes in other positions, the folding and trimerization of ha was generally tolerant of mutation of other tm residues to alanine. mutations in the region of ha predicted to be just outside and within the outer leaflet of the bilayer caused a loss of apical sorting and loss of insolubility in triton x-100. the effects of the mutations increased as the region mutated approached two tm residues at positions 520 and 521 that are highly conserved among influenza virus ha types (fig. 4), and decreased as the region mutated approached the portion of the tm that would reside in the inner leaflet of the bilayer. the pattern observed is consistent with ha having an important recognition feature within the membrane that contains s521 as the most important element. the observations that certain ha mutants not only lost the ability to be sorted, but were actually sorted to the basolateral surface as efficiently as the wild - type ha was sorted apically, could have two independent causes. either ha contains weak basolateral sorting information that is normally recessive to apical sorting information in the tm, which the mutant lacks, or ha must be able to enter digs to gain access to the apical surface, and the mutant can not enter digs. our data support the latter interpretation. decreasing cholesterol content in cells to the extent that twofold less ha partitioned into digs had no effect on sorting of wild - type ha, or a mutant of ha with a strong basolateral sorting signal. however, transport of the 2a520 mutant became random under the same conditions. these results suggest that the 2a520 mutant had no basolateral sorting information, but in the presence of digs was excluded from the apical pathway. under the conditions of our experiments, lowering cholesterol levels such that half as much wild - type ha was recovered in a cell fraction insoluble in triton x-100 did not decrease sorting of ha to the apical surface. recently, keller and simons (1998) reported that depleting cholesterol levels by 6070% caused missorting of ha to the basolateral surface of mdck cells infected with influenza virus. however, in our cell lines, ha expression is at least 50-fold lower than in cells infected with influenza virus and it is possible that sufficient apical sorting capacity remained intact under our experimental conditions to accommodate the available ha cargo. nonetheless, the fact that only half as much wild - type ha was found in the detergent - insoluble fraction but apical sorting was unchanged means that the sorting event is not identical to the partitioning event, as measured by this assay. in cholesterol - depleted cells, either the apical sorting machinery remained with the residual digs and was sufficient to sort has, or the machinery was able to interact with the apical signal in the tm of has outside of digs. the later possibility is supported by the observation that mutant a516s was as insoluble in triton x-100 as was wild - type ha, but was not sorted apically. thus, entry into detergent insoluble membranes is not sufficient for apical sorting of ha. a simple explanation for our observations is that apical sorting machinery is normally sequestered in digs. we propose that sequences including s521 form the binding site for an integral membrane protein associated with digs. the behavior of the ha tm mutants is consistent with a multistep process for sorting proteins into the apical pathway. in the first step, only proteins capable of entering membrane domains enriched in glycosphingolipids are able to come into contact with apical sorting machinery associated with those domains. however, only those proteins having the ability to bind tightly to elements of the sorting apparatus will be efficiently concentrated within the digs and be effectively sorted to the apical surface. the degree to which transmembrane proteins lacking sorting signals would reach the apical surface would depend upon their partitioning coefficient with digs, and upon the availability of empty space within the nascent apical transport vesicle. the latter would depend upon the concentration of cargo with apical sorting signals. all evidence suggests that the basolateral sorting machinery resides outside of digs. the probability that a protein would be transported basolaterally would depend upon the relative affinity of that protein for the basolateral sorting machinery, its partitioning coefficient with digs, and available space within the basolateral vesicles. our observations with the ha mutant 2a520 indicate that, under certain conditions, the inability to partition into digs may be sufficient to force a protein into the basolateral pathway. perhaps the converse is true, that efficient partitioning into digs might keep proteins from entering the basolateral pathway by default. it is possible that proteins with glycosphingolipid membrane anchors are sorted apically in such a manner, without interacting with the machinery that sorts apical transmembrane proteins. amino acids of the a / japan h2 ha tm are given in single letter code beginning with the last four residues of the external domain and ending with the residue predicted to be the last of the tm., no change from the wild - type sequence ;, deletion of the residue at that position. effect of mutations in the ha tm on kinetics of transport through the secretory pathway results are shown from a representative pulse - chase experiment on mdck cells permanently expressing various ha mutants. arrival at the golgi was monitored by the appearance of terminally glycosylated ha, which migrates more slowly during page. arrival at the cell surface was measured as the percentage of ha that was converted into its ha1 and ha2 subunits by trypsin added to the chase medium. mutations in the ha tm affect transport to the apical cell surface and solubility in triton x-100. (a) mdck monolayers expressing the ha types shown were grown on filter culture inserts for 5 d and then subjected to a pulse - chase protocol in which trypsin was present during the chase in either the apical (ap) or basolateral (bl) compartment. after the chase, has were immunoprecipitated, separated by page, and then analyzed by a phosphorimager. a representative image comparing ha and two mutants is shown. (b) mdck cells expressing the mutants shown were pulse - labeled and chased for 40 min, then the cells were lysed in 1% triton x-100 on ice. the cell lysate was centrifuged in a microfuge and separated into supernatant (s) and pellet (p) fractions. ha was immunoprecipitated from each fraction and analyzed as in a. effect of mutations in the ha transmembrane domain on the delivery of the protein to the apical surface of mdck cells results are shown from pulse - chase experiments on mdck cells permanently expressing various ha mutants. arrival at the apical cell surface was measured by adding trypsin to the chase medium on either the apical or basolateral side of the cell to convert ha into its ha1 and ha2 subunits. the percentage of ha arriving at the apical surface was calculated by dividing fraction of ha cleaved to ha1 and ha2 when trypsin was present at the apical side of the monolayer by the fraction cleaved by trypsin present on both sides of the monolayer. values are averages from three or more independent experiments and are presented with standard deviations. the fraction of each mutant that was insoluble when cells were lysed in triton x-100 was determined in parallel experiments. for three or more experiments depletion of cellular cholesterol allows expression of ha 2a520 to become more apical but has no effect on the distribution of wild - type ha or ha+8 d549h, a basolaterally expressed ha mutant cholesterol was depleted from mdck cell monolayers expressing each of the ha proteins by treatment overnight in cholesterol - free medium containing 50 m of compactin according to published methods (hua., 1996). distribution of has at the cell surface was measured as described for table ii. the thickness of the bar beneath the ha tm sequence is proportional to the severity of the inhibition of apical transport., positions of the sequence most conserved among influenza a and b has. transmembrane sequences from 36 ha proteins selected from all 14 influenza a subtypes and including three type b has were compared. for each position in the sequence of the tm, a conservation value was calculated by dividing the percentage of sequences having the most common amino acid by the number of different amino acids found at that position. for each position in the tm sequence, the conservation value and the most common amino acid are displayed. amino acids that would reside in the outer leaflet of the membrane bilayer are more conserved than those predicted to reside within the inner leaflet.	the composition of the plasma membrane domains of epithelial cells is maintained by biosynthetic pathways that can sort both proteins and lipids into transport vesicles destined for either the apical or basolateral surface. in mdck cells, the influenza virus hemagglutinin is sorted in the trans - golgi network into detergent - insoluble, glycosphingolipid - enriched membrane domains that are proposed to be necessary for sorting hemagglutinin to the apical cell surface. site- directed mutagenesis of the hemagglutinin transmembrane domain was used to test this proposal. the region of the transmembrane domain required for apical transport included the residues most conserved among hemagglutinin subtypes. several mutants were found to enter detergent - insoluble membranes but were not properly sorted. replacement of transmembrane residues 520 and 521 with alanines converted the 2a520 mutant hemagglutinin into a basolateral protein. depleting cell cholesterol reduced the ability of wild - type hemagglutinin to partition into detergent - insoluble membranes but had no effect on apical or basolateral sorting. in contrast, cholesterol depletion allowed random transport of the 2a520 mutant. the mutant appeared to lack sorting information but was prevented from reaching the apical surface when detergent - insoluble membranes were present. apical sorting of hemagglutinin may require binding of either protein or lipids at the middle of the transmembrane domain and this normally occurs in detergent - insoluble membrane domains. entry into these domains appears necessary, but not sufficient, for apical sorting.
dens invaginatus (di) is referred to as a developmental anomaly that results from an infolding within the crown prior to calcification. according to the 0.04 to 10% frequency of dens invagination in the currently available literature, several hypotheses have been proposed concerning the etiology of invaginated teeth, including constriction of the dental arch on the enamel organ, decreased or increased growth rate of the internal enamel epithelium, distortion of the enamel organ during dental development or insufficient nutrition of a portion of a single tooth germ. the epithelium forms a lining of enamel in a channel inside the dentin of the affected tooth. following calcification of the enamel, it radiographically appears as parallel radiopaque lines within the less radiopaque dentin. the enamel lining is sometimes imperfect and develops caries, which can lead to dentin or pulpal exposure and eventual pulpal pathosis. maxillary lateral incisors are most commonly affected and bilateral occurrence is not uncommon, involving 43% of all cases [6, 7 ]. dens evagination (de) (also known as talon cusp) is a relatively infrequent developmental abnormality characterized by the existence of an accessory cusp - like structure projecting from the cingulum area or cemento - enamel junction (cej) of the maxillary or mandibular anterior teeth both in the primary and permanent dentition [810 ]. de can occur in both sexes and the most commonly affected teeth are the permanent maxillary incisors, particularly the lateral. although both di and de have been reported frequently in the literature, simultaneous occurrence of these two entities within a single tooth has been noticed only limitedly [1315 ]. to the best of our knowledge, co - existence of a de with two dis within a single tooth is considered a rarity and has not been encountered in the literature. a 21-year - old girl came to the school of dentistry, kerman university of medical sciences for her regular dental follow - up visits and was advised to receive a full - mouth periapical radiographic evaluation. intra - oral examination revealed several carious lesions, restorations and a mild periodontal disease. presence of a de was noticeable in the patient s permanent right maxillary lateral incisor (fig 1). the patient s maxillary left lateral incisor did not have any problem based on clinical and radiographic evaluations. periapical radiographic evaluation showed co - existence of two dis with the aforementioned de (fig 2). the tooth responded as vital on electrical and thermal pulp sensitivity tests and no periapical lesion was detected radiographically. pits mesial and distal to the de were conservatively removed and restored by bonded restorations (3 m, espe, st paul, mn). dens invaginatus is a relatively common dental anomaly. it has been stated that di affects maxillary lateral incisors in 0.25% to 5.1% of the population [16, 17 ]. however, double di in a single tooth has infrequently been reported [14, 18, 19 ]. it is possible for dental caries to easily reach the pulp chamber in di cases. the patient is usually detected incidentally by intraoral periapical radiographs. the reported patient did not have any complaint about the considered tooth and the problem was accidentally diagnosed on full - mouth radiographic examination. according to oehlers description of invagination, the case was categorized as type i because the invaginated cavities did not extend beyond the cemento - enamel junction. it has been stated that the morphology of the tooth with di may undergo changes such as increased labio - lingual or mesio - distal dimension, incisal notching associated with a labial groove, a peg - shaped or conical morphology or the presence of an exaggerated palatal cingulum or talon cusp [21, 22].simultaneous occurrence of two dis with one de in a single tooth has not been previously reported but separate conditions in different teeth have been reported exclusively in the literature. in other words de is a rare dental anomaly occurring commonly on the lingual surface of primary or permanent teeth [23, 24 ]. usually, grooves or fissures can be found at the junction of the evagination with the lingual tooth surface, which often allows plaque retention, thereby causing caries and endodontic and periodontal inflammation. the etiology of this condition is multifactorial with genetic and environmental factors playing significant roles. it has also been reported that this anomaly is related to rubinstein - taybi, mohr, and sturge - weber syndromes and to anomalies including odontome, dens invaginatus, double tooth, supernumerary tooth, peg - shaped lateral incisors, agenesis of canines, mesiodens, megadont, and shovel - shaped incisors [8, 12, 24, 25, 27, 28 ]. although there is no strong correlation between the aforementioned syndromes and talon cusp, in a study by hennekam and van doorne on 45 patients with rubinstein - taybi syndrome, 92% of these patients had talon cusps. other signs of this syndrome such as mental and developmental retardation, thin upper lip, retrognathia, micrognathia and cleft palate were not evident in our patient, therefore the presence of this syndrome was ruled out. talon cusp has a prevalence of 0.067.7 percent with a predominance in the male gender [12,23, 30, 31 ]. although many authors have reported de in the primary dentition [8, 25 ], it is seen more frequently in permanent dentition. talon cusp can cause problems for the patient including poor esthetics, caries, occlusal trauma, displacement of the involved tooth, irritation of the tongue during speech and mastication, periodontal problems, accidental cuspal fracture and attrition causing pulpal exposure or periapical pathosis [11, 12, 3234 ]. the radiographic appearance of talon cusp resembles that of a supernumerary tooth or compound odontoma, consequently increasing the likelihood of misdiagnosis [11, 12, 32 ]. de can either be treated conservatively or radically, depending on the shape or size of the affected tooth [11, 28, 33, 3537 ]. treatment remedies may consist of gradual periodic cuspal reduction with fluoride as a desensitizing agent, one - visit reduction with or without endodontic therapy, application of sealants for developmental grooves and placement of esthetic restorations. in this case, simultaneous occurrence of two dis and one de was encountered in a permanent maxillary lateral incisor. this patient represented positive responses to vitality tests without any radiographic signs of apical periodontitis. in such cases, it is recommended that the susceptible parts should be sealed prophylactically using restorative materials in order to prevent formation of any communication pathways between the oral environment and pulpal space. it has to be taken into consideration that ohlers classification is based on the presence of a single dens invagination ; whereas, this patient represented two invaginations simultaneously. according to similar reports in the literature [18, 38 ], this classification should be modified based on the presence of multiple defects.	a case with two simultaneous dens invaginations (dis) and one dens evagination (de) in a permanent maxillary lateral incisor is reported for the first time in a 21-year - old girl.de known as talon cusp of the anterior teeth is a rare entity and its co - existence with di has been reported scarcely in the literature. simultaneous occurrence of two dis with one de has not been reported elsewhere. undoubtedly, familiarity with the internal anatomy of such a rare condition can help prevent pulpal disease while performing restorative procedures.
a 60-year - old man was referred to our outpatient clinic because of persistent air leakage lasting 17 days from his chest tube, which had been inserted for empyema at another hospital. he had a past history of diabetes mellitus and tuberculous pleuritis treated with antituberculous medicine 30 years ago. he had not been treated previously for empyema by drainage procedures such as closed thoracostomy, percutaneous catheter aspiration, or drainage. a chest computed tomography (ct) scan showed loculated pleural fluid collection with air density and pleural wall thickening with enhancement and calcification. further, a 2.2-cm mass - like lesion adjacent to the margin of an empyema cavity was identified retrospectively (fig. laboratory tests revealed a slightly elevated c - reactive protein level of 1.0 mg / dl, while other parameters were within the normal range. seven days after admission, we planned decortication because of prolonged air leakage and the patient s desire for surgery. the fibrinous contents of the empyema cavity were removed, and the lung was decorticated without difficulty. therefore, the operation was completed after the confirmation of the full expansion of the lung. contrary to our expectations, however, the histopathological diagnosis after the operation was malignant lymphoma. immunohistochemically, the tumor cells were positive for leukocyte common antigen, cd79a, ki-67 (range, 7080%), and cd3, and negative for cd20. 2). after the identification of a monoclonal proliferation of b - cells by an immunoglobulin heavy- chain gene rearrangement, the final diagnosis of diffuse large b - cell lymphoma with an aberrant expression of a t - cell marker was made. the postoperative period was uneventful, and the chest tube was removed on postoperative day 12. we considered the possibility of incomplete resection, and the patient was referred to the hematology department for adjuvant chemotherapy. pyothorax - associated lymphoma (pal) is a rare malignant lymphoma developing in the pleural cavity after long - standing pyothorax. according to the current world health organization histological classification published in 2005, pal is defined as a neoplasm of large b - cells, typically with immunoblastic morphology, usually presenting as a pleural mass. it occurs in patients with a history of long - standing pyothorax resulting from pulmonary tuberculosis or tuberculous pleuritis and is strongly associated with the epstein barr virus (ebv). a majority of these cases have been reported in japan, although some cases have occurred in western countries. it is assumed that the higher prevalence in japan is caused by a higher incidence of ebv infection and lung collapse therapy for tuberculosis in asia, particularly in japan. the etiology of pal is not clearly understood. however, previous reports have suggested that artificial pneumothorax, ebv latent infection, cytokines such as interleukin-6 and -10, and oxidative stress produced during chronic inflammation might be important factors for pal development. reported a summary of clinical and pathological findings in 106 patients with pal in japan. the median age of the patients was 64 years (range, 46 to 82 years) with a male / female ratio of 12.3:1. the interval between the onset of pleuritis and the initial symptoms of lymphoma was 37 years (range, 20 to 64 years). all of the cases were of non - hodgkin s lymphoma, among which the diffuse large b - cell type was the most common (88%). the definitive diagnosis of pal can be made by histopathological and immunohistochemical examinations of biopsy or surgically resected specimens. a typical histological examination demonstrates a diffuse destructive proliferation of large cells with a predominant population of immunoblasts. lymphoma cells are mostly positive for cd20 and cd79a in the immunohistochemical examination. according to the pathological findings by aozasa., a majority of the cases were cd20 + and/or mbi+, cd45ro-, and cd3-, and were of the b - cell lineage. however, there could be an aberrant phenotype with the expression of some t - cell markers, as in our case. ueda. analyzed the radiological features of pal to help in the diagnosis of this rare malignant lesion. they reported that a typical radiological finding of pal was a pleural soft - tissue mass adjacent to the margin of a coexistent empyema cavity and the shape of the mass demonstrated on the ct scan was mostly lenticular or crescentic. we were able to find a mass - like lesion on the ct scan retrospectively by reviewing the report of ueda. although the optimal treatment is not well - established, most patients with pal have received chemotherapy and/or radiotherapy, as reported in the literature. the prognosis of pal reported in 2002 is poor, with a five - year survival rate of 21.6%, although a better survival rate is observed in patients who are responsive to chemotherapy. however, a recent survey reported an improved overall five - year survival rate of 35% and suggested several prognostic factors for the overall survival. in summary, we have reported a case of pal of a b - cell origin resulting from tuberculous pleuritis. although pal is a relatively rare disease, it should be considered a potential diagnosis in patients with a history of chronic pyothorax.	pyothorax - associated lymphoma is a relatively rare type of lymphoma that occurs in patients who have long histories of tuberculous pleuritis or induced pneumothorax. it is a type of non - hodgkin s lymphoma of mainly the b - cell phenotype and is strongly associated with epstein barr virus infection. a majority of these cases have been reported in japan, although some cases have occurred in western countries. here, we describe a case of pyothorax - associated lymphoma in a patient with a 30-year history of chronic tuberculous empyema. the patient underwent decortication under the impression of chronic empyema with fistula. the histopathologic diagnosis was a diffuse large b - cell lymphoma associated chronic inflammation.

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